Download Gene Detection Systems Catalog

2005 Edition
Non-Radioactive Gene Detection Systems,
Triple Repeat Disorders, Mutation and Gene Detection,
Control DNA’s,
Gene Detection Systems & Reagents
GeneProber™ Southern based Non-Radioactive Gene Detection System
PCRProber™ & Genemer™ PCR based Gene Detection System
GScan™ Fluorescent Gene Detection System
Genemer™ Control DNA
Reagents & Tools
“Quality, Consistency, Confidence”
Gene Link has acquired a reputation for quality, consistency and confidence in
the critical gene research tools and technology we supply to the research
community worldwide. Our products and services are supported and ensured by
our commitment to premium quality and our constant efforts to introduce new
and improved products.
Gene Link fosters customer satisfaction and loyalty by maintaining personal
relationships with our customers. We routinely assist our customers regarding
the technical inquiries, the design of their experiments and the solving of their
application problems. Our dedicated and highly trained staff of customer service
and technical support employees is strongly motivated to serve our customers.
Gene Link has endeavored to develop, and will continue to preserve, our
reputation for “Quality, Consistency, and Confidence.”
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Table of Contents
General Information
About Us
Confidentiality/ Nondisclosure Agreement
General Information
Ordering Information
Terms and Conditions of Sale
5
7
8
9
10
11
Molecular Analysis of Genetic Diseases
13
Gene Link Gene Detection Systems
Introduction
Gene Detection Systems
Fragile X Syndrome
Huntington’s Disease
Myotonic Dystrophy
Friedreich’s Ataxia
Kennedy Disease
RhD (RhD gene exon 10 specific)
Rh EeCc (Rh Ee and Cc exon 7 specific)
SRY (sex determining region on Y)
Sickle Cell
Cystic Fibrosis (various mutations)
Tay Sach’s Syndrome
Gaucher’s Disease
27
29
29
31
39
49
57
65
71
71
75
81
85
89
93
Gene Detection Systems Product Line
GeneProber™
GeneProber™ Digoxigenin Labeled Probes
PCRProber™ Gene Detection Kits
PCRProber ™ Alkaline Phosphatase Labeled Probes
Genemer™
Genemer™ Kits
Genemer™ Radioactive Detection Kits
GScan™ Gene Detection Kits
GScan™ Genemer Controls
Genemer™ Control DNA
97
100
100
101
101
102
103
104
105
106
107
Genetic Tools and Reagents
109
Appendix
123
Protocols
141
Genetic Glossary
161
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General Information
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About Us
Gene Link, Inc. is a dynamic biotechnology company and research organization.
Our mission is to be one of the most reliable suppliers of reagents and reagent
systems used in genetic research technology applications worldwide.
Gene Link, Inc. is privately held biotechnology company incorporated in the
State of New York dedicated to fundamental and applied research on
genetic/biomedical projects relating to gene mapping, localization, detection and
marketing of our services and products in these areas. Since inception, Gene
Link has acquired a reputation for quality, consistency and confidence in the
services and products it provides to the research communities worldwide. Our
services and products are supported and ensured by our commitment and
constant efforts to excel. Gene Link is situated in Westchester County of New
York, a location ideally suited for growth and strong interaction with prestigious
nearby universities, biotechnology and pharmaceutical companies in New York,
New Jersey and Connecticut.
Gene Link fosters customer satisfaction and loyalty by maintaining close
personal relationships with our customers, and by offering technical support for
the applications of our products. We routinely assist our customers regarding the
design of their experiments and the solving of their application problems to help
researchers reach their goals faster and with greater success. Gene Link
customers are Universities, Research Institutes, Pharmaceutical companies and
Hospitals involved in research. Understanding the needs of our customers and
helping them achieve success is a priority at Gene Link. These customers are
located worldwide.
Gene Link is a leading supplier of custom oligonucleotides for use in PCR,
sequencing, cloning, and mutagenesis. Gene Link services include genotyping,
sequencing and gene construction. Gene Link offers as well a wide variety of
other molecular biology products such as, siRNA, fluorescent probes and nonradioactive gene detection systems for human genetic disorders.
Gene Link's research and development activities focus on developing novel gene
detection systems. Gene Link is in the process of developing research products
to aid scientists with detection of genetic disorders, specifically triple repeat
disorders (Fragile X, Myotonic Dystrophy, Huntington etc.). Also available from
Gene Link are kits for research purposes designed for genotyping other genetic
disorders. To complement our genetic disorder detection systems we have
recently introduced detection systems for infectious disorders.
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Confidentiality
Nondisclosure Agreement
At Gene Link, we respect your privacy and are committed to protecting it. We are committed to building customer trust by
demonstrating this respect in every aspect of our marketing activities. If at any time you feel that Gene Link is not following
its stated privacy policy, please feel free to contact us at 1-800-GENE-LINK with your concerns. The points below summarize
our promise to you.
•
The information you provide to Gene Link through our website or any other channel will be used only to provide you
with communications that are relevant to Gene Link products and services that we feel may suit your needs and
preferences.
•
This information is held in strict confidence and will never be sold, traded or rented to other companies, individuals
or entities for their marketing use.
•
Every communication message that you receive from Gene Link will provide you with the opportunity to be removed
from future information exchanges.
•
You may also send an email to [email protected] to request removal from our email or postal mail list.
Please type the word "REMOVE" in the subject line and include your name, company name and the list(s) from which
you would like to be removed.
•
Upon request, we will provide you with a summary of the information you have provided to Gene Link. This
information will be provided only to the email address on file for the customer making the request. You will be able
to change, correct or remove any information at any time. The contact for such requests is
[email protected]
•
Following website registration our site will use "session cookies" to help provide relevant information to you during
your visit. The cookies are only accessible by Gene Link while you are visiting our site and are deleted upon your
signoff.
•
You may terminate your website registration at any time by sending an email to [email protected]
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General Information
Products
Gene Link is committed to providing the highest quality products at competitive prices. Gene Link warrants that all products
meet or exceed the performance standards described in the product specification sheets. If you are not completely satisfied
with our product, our policy is to replace the replace the product as soon as possible. Gene Link provides no other warranties
of any kind, expressed or implied. Gene Link’s liability shall not exceed the purchase price of the product. Gene Link shall
have no liability for direct, indirect, consequential or incidental damages arising from the use, results of use, or inability to use
its products.
•
•
•
•
•
•
•
Privacy
At Gene Link, we respect your privacy and are committed to protecting it. We are committed to building customer trust by
demonstrating this respect in every aspect of our marketing activities. If at any time you feel that Gene Link is not following
its stated privacy policy, please feel free to contact us at 1-800-GENE-LINK with your concerns. The points below summarize
our promise to you.
The information you provide to Gene Link through our website or any other channel will be used only to provide you with
communications that are relevant to your needs and preferences.
This information is held in strict confidence and will never be sold, traded or rented to other companies, individuals or entities
for their marketing use.
Every communication message that you receive from Gene Link will provide you with the opportunity to be removed from
future information exchanges.
You may also send an email to [email protected] to request removal from our email or postal mail list. Please type
the word "REMOVE" in the subject line and include your name, company name and the list(s) from which you would like to be
removed.
Upon request, we will provide you with a summary of the information you have provided to Gene Link. This information will be
provided only to the email address on file for the customer making the request. You will be able to change, correct or remove
any information at any time. The contact for such requests is [email protected]
Following website registration our site will use "session cookies" to help provide relevant information to you during your visit.
The cookies are only accessible by Gene Link while you are visiting our site and are deleted upon your signoff.
You may terminate your website registration at any time by sending an email to [email protected]
Customer Service
Customer Service representatives are available to Monday through Friday from 9:00 AM to 6:00 PM Eastern Time. E-mail:
[email protected]. Gene Link does not require written confirmation for telephone, e-mail or Internet orders. To
avoid duplication, be certain that any written confirmation of an order is clearly marked CONFIRMING.
Payment
Payment terms are net 30 days.
Credit Cards
Gene Link accepts credit card payments (Visa, MasterCard and American Express only via phone, fax, and mail). Please
provide card number, expiration date, and card billing name and address.
Shipping
In stock catalog items regularly ship same day of order receipt for next day delivery. All custom oligos are regularly shipped
in 24 hrs. and guaranteed to be shipped within 48 hrs. of order receipt by Standard Overnight for guaranteed afternoon
delivery. Upon request, Priority Overnight shipment will be made for guaranteed morning delivery. Exceptions may be
necessary for US holidays. A $10.00 handling charge is added to each order in addition to shipping charges. Please consult
Customer Service for additional shipping information.
Blanket Orders
As a service to our customers, Gene Link accepts blanket order. For your convenience, we encourage you to use this type of
purchase order. For more information please contact your Purchasing department or Customer Service department.
Returns
Products may not be returned without proper authorization by the Gene Link Customer Service Department. Due to the
custom nature and temperature sensitivity of most our products, we are unable to restock and resell returned goods.
Intended Use
All products sold by Gene Link are intended for research only. These products are not suitable for diagnostic or drug purposes,
nor are they suitable for administration to humans or animals.
International Orders
Gene Link has Distributors worldwide. Please see inside back cover to find a distributor in your area.
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Ordering Information
All customers are encouraged to place orders through our easy to use on-line ordering system at www.genelink.com
Registration and Login
• Existing Customers
All existing customers have a Gene Link assigned customer number. This Customer Number is also referred to as the Customer ID.
The Gene Link assigned customer number should be used for all logins and is the password as default. Password is required for
order tracking and online catalog products orders. This requirement is only for online catalog item products and for checking order
history and status. Password is not required for custom oligo orders.
• New Customers
All new customers can place the first order without having a customer number. At Gene Link customers are assigned a number after
their first order is received. All new customers are requested to register and provide all the requested information. The order will be
processed and you will be contacted if any more information is required. A Gene Link assigned customer number will be emailed to
you for future use.
• Individual Users
At Gene Link 'Customer Numbers' are assigned to Principal Investigators, Laboratory Directors or Independent Laboratories.
Researchers within the same 'laboratory' use the same Customer Number. Individual researchers are termed as 'User' and
registered. All orders placed are thus linked to a customer number and a user. A new researcher in a laboratory registers under the
'Customer Number' account.
Password Requirement
Our new web site offers the security of password protection for placing catalog orders and viewing order status and history. Customers
can feel confident that their information is protected, while enjoying the convenience of acquiring account information immediately, at
the click of a mouse. As a default, your password will be the same as your customer number. You will be able to change it when you
log on. Please make sure that all parties ordering for your account are aware of the password. We advise that password be changed
only after consultation with all 'users' of the account, specifically after approval by of the Laboratory Director or Principal Investigator.
Forgot Password: Password will be emailed to the email address in the 'Ship To' information of the Account Profile. The email
address entered for the 'email password' should exactly match the one on file.
Catalog Product Orders
Select the 'catalog orders' button to browse or place catalog orders. Select products to order and view their specification sheets. To
finally submit order after viewing and approving the items on the cart you will require a Gene Link assigned Customer Number or new
customers simply register as a 'new customer'.
Order Confirmation
All submitted online orders receive a message indicating the order number and the email address where the automatic email
confirmation will be sent. Please print or make a note of this information. The email confirmation is an official record of order placed
and should be saved for future reference. The email address used for sending the automatic confirmation is of the 'User'. Please select
your name from the dropdown USER name field. If you are a 'new' user, select NEW and enter your name, email address and
telephone number. This information will be stored and your name will appear the next time you login.
Order Changes and Cancellation
All catalog product orders are routinely shipped the same day for next day delivery. Please email us as soon as possible if you notice
an error or want to amend an order. Reference the order number in all correspondence with Gene Link. The email address for
immediate inquiries relating to the order on the day it was placed is [email protected] All inquiries after 24 hrs. of order
placement may please be addressed to [email protected].
Order Tracking and Order History
Order Tracking and Order History
All orders are assigned an order number once placed. An email confirmation of all orders contains the order number. You can track the
shipment status of orders online by clicking the 'Order Status' menu item on the menu bar. You will be required to enter your
customer number and password to access order status and order history.
Orders by Phone: 1-800-GENE-LINK
Orders by Fax:
1-888-GENE-LINK
(Please note that custom oligo orders cannot be placed by phone)
Orders by E-mail: [email protected]
When placing an order, please provide the following information:
1.
Purchase order number or credit card number
2.
Customer number
3.
Billing address
4.
Shipping address
5.
Name of person to whose attention order should be shipped
6.
Name and telephone number of contact person
7.
Product catalog number, description, size and quantity
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Terms and Conditions of Sale
1. Acceptance – GENE LINK, Inc., HERIN REFERRED TO AS SELLER, RESERVES THE RIGHT TO ACCEPT OR REJECT A SALES ORDER AT ITS
SOLE DISCRETION WITHOUT ASSIGNING ANY REASON. ALL SALES ARE SUBJECT TO AND EXPRESSLY CONDITIONED UPON THE TERMS AND
CONDITIONS CONTAINED HEREIN, AND UPON BUYER'S ASSENT THERETO. NO VARIATION OF THESE TERMS AND CONDITIONS WILL BE
BINDING UPON SELLER UNLESS AGREED TO IN WRITING AND SIGNED BY AN OFFICER OR OTHER AUTHORIZED REPRESENTATIVE OF SELLER.
2. Changes -Orders arising hereunder may be changed or amended only by written agreement signed by both Buyer and Seller, setting forth
the particular changes to be made and the effect, if any, of such changes on the price and time of delivery. Buyer may not cancel this order
unless such cancellation is expressly agreed to in writing by Seller. In such event, Seller will advise Buyer of the total charge for such
cancellation, and Buyer agrees to pay such charges, including, but not limited to, storage and shipment costs, costs of producing non-standard
materials, costs of purchasing non-returnable materials, cancellation costs imposed on Seller by its suppliers, and any other cost resulting from
cancellation of this order by Buyer which is permitted by Seller. Certification of such costs by Seller's independent public accountants shall be
conclusive on the parties hereto.
3. Delivery, claims, delays - All sales are FOB Seller's shipping point unless otherwise noted. If Shipping and Handling Charges are quoted or
invoiced, they will include charges in addition to actual freight costs. Delivery of the goods to the carrier at Seller's shipping point shall
constitute delivery to Buyer but Seller shall bear all risk of loss or damage in transit. The general method of shipment for each item is Airborne
Express next afternoon service unless otherwise specified. However, Seller reserves the right, in its discretion, to determine the exact method
of shipment. Seller reserves the right to make delivery in installments, all such installments to be separately invoiced and paid for when due per
invoice, without regard to subsequent deliveries. Delay in delivery of any installment shall not relieve Buyer of Buyer's obligations to accept
remaining deliveries.
Immediately upon Buyer's receipt of any goods shipped hereunder, Buyer shall inspect the same and shall notify Seller in writing of any claims
for shortages, defects or damages and shall hold the goods for Seller's written instructions concerning disposition. If Buyer shall fail to so notify
Seller within five days after the goods have been received by Buyer, such goods shall conclusively be deemed to conform to the terms and
conditions hereof and to have been irrevocably accepted by the Buyer.
Seller shall not be liable for any loss, damage or penalty as a result of any delay in or failure to manufacture, deliver or otherwise perform
hereunder due to any cause beyond Seller's reasonable control, including, without limitation, unsuccessful reactions, act of Buyer, embargo or
other governmental act, regulation or request affecting the conduct of Seller's business, fire, explosion, accident, theft, vandalism, riot, acts of
war, strikes or other labor difficulties, lightning, flood, windstorm or other acts of God, delay in transportation, or inability to obtain necessary
labor, fuel, materials, supplies or power at current prices.
4. Allocation of goods - If Seller is unable for any reason to supply the total demands for goods specified in Buyer's order, Seller may allocate
its available supply among any or all Buyers on such basis as Seller may deem fair and practical, without liability for any failure of performance
which may result there from.
5. Payment - Terms of sale are net 30 days of date of invoice, unless otherwise stated. If the financial condition of Buyer results in the
insecurity of Seller, in its sole and unfettered discretion, as to the ultimate collectability of the purchase price, Seller may, without notice to
Buyer, delay or postpone the delivery of the products; and Seller, at its option, is authorized to change the terms of payment to payment in full
or in part in advance of shipment of the entire undelivered balance of said products. In the event of default by Buyer in the payment of the
purchase price or otherwise, of this or any other order, Seller, at its option, without prejudice to any other of Seller's lawful remedies, may
defer delivery, cancel this Contract, or sell any undelivered products on hand for the account of Buyer and apply such proceeds as a credit,
without set-off or deduction of any kind, against the contract purchase price, and Buyer agrees to pay the balance then due to Seller on
demand. Buyer agrees to pay all costs, including, but not limited to, reasonable attorney and accounting fees and other expenses of collection
resulting from any default by Buyer in any of the terms hereof.
6. Taxes and other charges - Any use tax, sales tax, excise tax, duty, custom, inspection or testing fee, or any other tax, fee or charge of
any nature whatsoever imposed by any governmental authority, on or measured by the transaction between Seller and Buyer shall be paid by
Buyer in addition to the prices quoted or invoiced. In the event Seller is required to pay any such tax, fee or charge, Buyer shall reimburse
Seller therefore; or, in lieu of such payment, Buyer shall provide Seller at the time the order is submitted an exemption certificate or other
document acceptable to the authority imposing the tax, fee or charge.
7. Pricing - Prices are subject to change without notice. Please inquire about volume discounts. Please call us for current prices if you require
this information prior to placing your order. We guarantee our written domestic quotations for one (1) year. For guarantee information
regarding quotations outside the US, please contact our distributor in your local area. When placing your order, please reference our quoted
prices or our pro forma number. If you place your order by phone, we will confirm our current price at that time.
8. Price Changes - Shipment will be made promptly even if prices have been nominally increased. Price reductions will be automatically
applied to your invoice.
9. Warranties - Seller warrants that its products shall conform to the description of such products as provided to Buyer by Seller through
Seller's catalog, analytical data or other literature. THIS WARRANTY IS EXCLUSIVE, AND SELLER MAKES NO OTHER WARRANTY,
EXPRESS OR IMPLIED, INCLUDING ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR ANY PARTICULAR
PURPOSE. Seller's warranties made in connection with this sale shall not be effective if Seller has determined, in its sole discretion, that Buyer
has misused the products in any manner, has failed to use the products in accordance with industry standards and practices, or has failed to
use the products in accordance with instructions, if any, furnished by Seller.
Seller's sole and exclusive liability and Buyer's exclusive remedy with respect to products proved to Seller's satisfaction to be defective or
nonconforming shall be replacement of such products without charge or refund of the purchase price, in Seller's sole discretion, upon the return
of such products in accordance with Seller's instructions. SELLER SHALL NOT IN ANY EVENT BE LIABLE FOR INCIDENTAL, CONSEQUENTIAL OR
SPECIAL DAMAGES OF ANY KIND RESULTING FROM ANY USE OR FAILURE OF THE PRODUCTS, EVEN IF SELLER HAS BEEN ADVISED OF THE
POSSIBILITY OF SUCH DAMAGE INCLUDING, WITHOUT LIMITATION, LIABILITY FOR LOSS OF USE, LOSS OF WORK IN PROGRESS, DOWN
TIME, LOSS OF REVENUE OR PROFITS, FAILURE TO REALIZE SAVINGS, LOSS OF PRODUCTS OF BUYER OR OTHER USE OR ANY LIABILITY OF
BUYER TO A THIRD PARTY ON ACCOUNT OF SUCH LOSS, OR FOR ANY LABOR OR ANY OTHER EXPENSE, DAMAGE OR LOSS OCCASIONED BY
SUCH PRODUCT INCLUDING PERSONAL INJURY OR PROPERTY DAMAGE UNLESS SUCH PERSONAL INJURY OR PROPERTY DAMAGE IS CAUSED
BY SELLER'S GROSS NEGLIGENCE. All claims must be brought within one (1) year of shipment, regardless of their nature.
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10. Compliance with laws, regulations - Seller certifies that to the best of its knowledge its products are produced in compliance with
applicable requirements of the Fair Labor Standards Act, as amended, and the Occupational Safety and Health Standards Act of 1970 and
regulations, rules and orders issued pursuant thereto.
11. Buyer's use of products - Seller's products are intended primarily for laboratory research purposes and, unless otherwise stated on
product labels, in Seller's catalog or in other literature furnished to Buyer, are not to be used for any other purposes, including but not limited
to, in vitro diagnostic purposes, in foods, drugs, medical devices or cosmetics for humans or animals or for commercial purposes. Buyer
acknowledges that the products have not been tested by Seller for safety and efficacy in food, drug, medical device, cosmetic, commercial or
any other use, unless otherwise stated in Seller's literature furnished to Buyer. Buyer expressly represents and warrants to Seller that Buyer
will properly test, use, manufacture and market any products purchased from Seller and/or materials produced with products purchased from
Seller in accordance with the practices of a reasonable person who is an expert in the field and in strict compliance with all applicable laws and
regulations, now and hereinafter enacted. Buyer further warrants to Seller that any material produced with products from Seller shall not be
adulterated or misbranded within the meaning of the Federal Food, Drug and Cosmetic Act and shall not be materials which may not, under
Sections 404, 505, or 512 of the Act, be introduced into interstate commerce.
Buyer realizes that, since Seller's products are, unless otherwise stated, intended primarily for research purposes, they may not be on the Toxic
Substances Control Act (TSCA) inventory. Buyer assumes responsibility to assure that the products purchased from Seller are approved for use
under TSCA, if applicable.
Buyer has the responsibility to verify the hazards and to conduct any further research necessary to learn the hazards involved in using products
purchased from Seller. Buyer also has the duty to warn Buyer's customers and any auxiliary personnel (such as freight handlers, etc.) of any
risks involved in using or handling the products. Buyer agrees to comply with instructions, if any, furnished by Seller relating to the use of the
products and not misuse the products in any manner. If the products purchased from Seller are to be repackaged, relabeled or used as starting
material or components of other products, Buyer will verify Seller's assay of the products. No products purchased from Seller shall, unless
otherwise stated, be considered to be foods, drugs, medical devices or cosmetics.
12. Buyer's Representations and Indemnity - Buyer represents and warrants that it shall use all products ordered herein in accordance
with Paragraph No. 9 "Buyer's Use of Products", and that any such use of products will not violate any law or regulation. Buyer agrees to
indemnify and hold harmless Seller, its employees, agents, successors, officers, and assigns, from and against any suits, losses, claims,
demands, liabilities, costs and expenses (including attorney and accounting fees) that Seller may sustain or incur as a result of any claim
against Seller based upon negligence, breach of warranty, strict liability in tort, contract, or any other theory of law brought by Buyer, its
officers, agents, employees, successors or assigns, by Buyer's customers, by end users, by auxiliary personnel (such as freight handlers, etc.)
or by other third parties, arising out of, directly or indirectly, the use of Seller's products, or by reason of Buyer's failure to perform its
obligations contained herein. Buyer shall notify Seller in writing within fifteen (15) days of Buyer's receipt of knowledge of any accident, or
incident involving Seller's products which results in personal injury or damage to property, and Buyer shall fully cooperate with Seller in the
investigation and determination of the cause of such accident and shall make available to Seller all statements, reports and tests made by
Buyer or made available to Buyer by others. The furnishing of such information to Seller and any investigation by Seller of such information or
incident report shall not in any way constitute any assumption of any liability for such accident or incident by Seller.
13. Patent disclaimer - Seller does not warrant that the use or sale of the products delivered under will not infringe the claims of any United
States or other patents covering the product itself or the use thereof in combination with other products or in the operation of any process.
14. Returns - Goods may not be returned for credit except with Seller's permission, and then only in strict compliance with Seller's return
shipment instructions. Any returned items may be subject to a 20% processing fee.
15. Technical Assistance - At Buyer's request, Seller may, at Seller's discretion, furnish technical assistance and information with respect to
Seller's products. SELLER MAKES NO WARRANTIES OF ANY KIND OR NATURE, EXPRESS OR IMPLIED, INCLUDING ANY IMPLIED WARRANTY OF
MERCHANTABILITY OR FITNESS FOR ANY PARTICULAR PURPOSE, WITH RESPECT TO TECHNICAL ASSISTANCE OR INFORMATION PROVIDED BY
SELLER OR SELLER'S PERSONNEL. ANY SUGGESTIONS BY SELLER REGARDING USE, SELECTION, APPLICATION OR SUITABILITY OF THE
PRODUCTS SHALL NOT BE CONSTRUED AS AN EXPRESS WARRANTY UNLESS SPECIFICALLY DESIGNATED AS SUCH IN A WRITING SIGNED BY
AN OFFICER OR OTHER AUTHORIZED REPRESENTATIVE OF SELLER.
16. Miscellaneous - Seller's failure to strictly enforce any term or condition of this order or to exercise any right arising hereunder shall not
constitute a waiver of Seller's right to strictly enforce such terms or conditions or exercise such right thereafter. All rights and remedies under
this order are cumulative and are in addition to any other rights and remedies Seller may have at law or in equity. Any waiver of a default by
Buyer hereunder shall be in writing and shall not operate as a waiver of any other default or of the same default thereafter.
If any provision of this Agreement shall be held to be invalid, illegal or unenforceable, the validity, legality and enforceability of the remaining
provisions shall not be affected or impaired thereby. The paragraph headings herein are for convenience only; they form no part of the terms
and conditions and shall not affect their interpretation.
This Agreement shall be binding upon, inure to the benefit of, and be enforceable by, the parties hereto, and their respective heirs, personal
representatives, successors and assigns.
17. Governing Law - All disputes as to the legality, interpretation, application, or performance of this order or any of its terms and conditions
shall be governed by the laws of the State of New York including its conflict of laws principles. Each party to this order agrees that any dispute
arising between them, which results in either party instituting court proceedings shall be litigated in either the Federal District Court for the New
York area or in the Circuit Court for the City of Hawthorne.
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Molecular Analysis of Genetic Diseases: An Overview
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Molecular Analysis of Genetic Diseases: An Overview
Introduction
Our present understanding of the molecular basis of genetic disorders is principally due to the recent clinical advances in
recombinant DNA techniques. The revolution in development and implementation is now almost two decades in the making.
Most of the molecular genetic techniques now in clinical use were previously limited to laboratories involved in basic research;
currently, however, they are routinely available in service laboratories performing DNA diagnostics. This transition itselfmolecular technology from pure research to DNA service laboratories-is an extraordinary example of the impact of molecular
genetics. As a result of these advances, it is imperative that physicians and other health care professionals who provide the
clinical bridge between patient and laboratory possess a sound understanding of molecular diagnostic techniques.
The revolution in recombinant DNA technology has improved our understanding of simple mutations as causes of disease. The
molecular basis of genetic disorders is as varied as clinical medicine itself. The molecular etiology of disorders may be
fundamentally straightforward, such as in sickle cell disease, which is the best understood and the first disease whose
mutation was established at the DNA level. On the other hand, molecular genetics has delineated a whole new class of
disease where anticipation is involved; that is, the phenomenon of apparently increasing disease severity in successive
generations. Addressing the etiology of the more complex disorders that involve anticipation (e.g., fragile X syndrome,
myotonic dystrophy, and Huntington’s disease) is often challenging. One must consider such variables as paternal versus
maternal transmission of the mutant allele and the existence of additional factors, as noted in the association of Apo E4 allele
with late-onset Alzheimer’s disease.
Finally, the field of molecular genetics has been the motivating factor for approaches to molecular medical practice. Molecular
technologies have propelled diagnostic medicine from a predominantly clinical specialty-dysmorphology-where physical
diagnosis is key to direct evaluation of DNA and RNA and its impact on protein expression. In part, the Human Genome
Project serves as a framework for the construction of these advances and holds the promise of unraveling the mystery of
human genetics. Ultimately, the real achievements from this project will be the prevention and treatment of human genetic
afflictions. None of this will be accomplished without the support of the medical community and the public; to do this, one
must first appreciate the process of testing for diseases with a molecular basis.
This review provides an avenue to greater understanding of these exciting developments. The recombinant DNA techniques
presently used for the analysis of mutations are briefly explained. We present clinical examples as an introduction to the
techniques most commonly employed in service laboratories: direct detection assays, where specific mutation is recognized,
and indirect detection assays, useful for the deduction of an inheritance pattern where the actual mutation or its gene is not
known but may be closely linked to known DNA polymorphisms.
Human Genome, Polymorphisms, and Mutations
The human genome is generally considered to consist of about 3 x 109 base pairs (bp) per haploid chromosomal complement,
i.e., nearly 6 x 109 bp per diploid cell, encoded within the 22 autosomes, the X and Y sex chromosomes, and the mitochondrial
genome. Just a glance at the human population makes it evident that a wide range of phenotypic variation exists between
various races, ethnic groups, and other isolated groups. The human population is truly heterogeneous. These variations
comprise not only phenotypic, but also genotypic differences- differences at the DNA level. For example, the wide variation of
genotypes within blood groups ABO, MNSs, Rh, and the various forms (alleles) of other erythrocyte proteins have long been
established.
The molecular variations that result in protein differences are encoded at the DNA level and can be inherited. Variations such
as these within normal genes and proteins (which are, parenthetically, not deleterious) are termed polymorphisms. In other
words, polymorphisms represent two or more forms of functionally similar yet genetically distinct and structurally different
forms of DNA. A specific chromosomal site (locus) is said to be polymorphic when there are two or more alleles with a
frequency in the population of 1 % or greater. Those permanent changes in the DNA sequence that result in a disordered
(disease) state are known as mutations (although, in the strictest sense, any permanent change in DNA sequence may
correctly be termed a mutation); a mutation not causing a change in functional properties is termed a silent mutation and is
equivalent to a polymorphism. Polymorphisms have been immensely valuable as genetic markers for gene mapping by
linkage analysis and are used to both trace and deduce different inherited forms of alleles/genes in family studies.
The primary causes of changes in DNA sequence are random error during DNA replication and the effects of environmental
mutagens. The entire human genome is copied by replication during each cell division. It is surprising, therefore, that the end
result of replication is relatively error free; in fact, virtually all (>99.9%) DNA replication errors are corrected by the
"proofreading" property of DNA polymerases. Overall, the replication error is 10-10 per bp per cell division. Since the human
diploid genome contains approximately 6 X 109 bp of DNA, new mutations introduced per cell division due to replication error
are less than 1 new base pair mutation per cell division.
During an adult's lifetime, there are approximately 1015 cell divisions; thus, thousands of new mutations could conceivably
occur at nearly every base pair level of the DNA sequence. Fortunately, most mutations occur in somatic cells and are not
inherited, as they would be in germ cells (sperm or egg). Only germ line mutations are passed on from generation to
generation and are the basis of inherited disorders. Somatic cell mutations, although not inherited, may cause disease (e.g.,
cancer), depending on the type of mutation of a particular gene in a particular tissue. Understanding genetic polymorphism,
mutation, and mutation rates helps us appreciate the existence of our molecular heterogeneity and allows us to examine the
molecular approaches available to analyze the population variations.
- 15 -
Direct and Indirect Analysis Methods
The choice of using direct methods for mutation analysis is restricted by our knowledge of the existence of a mutation and its
type. The use of direct methods is clearly limited to the analysis of mutations that have previously been documented. In
almost all cases, the gene responsible for a particular abnormality has already been identified. Sickle cell disease is an
excellent example of direct DNA testing success; a discreet mutation is recognized and may be easily tested for in patients at
risk, with virtually a 100% rate of detection. Direct methods cannot be used for disorders for which the gene has not been
identified; instead, linkage with polymorphic markers is used to deduce the inheritance pattern of a particular allele.
Until recently, linkage analysis was the sole technique for detecting most genetic disorders (e.g., Huntington's disease, cystic
fibrosis, and myotonic dystrophy). On identification of a particular gene and etiologic mutations, direct analysis for each
mutation can be performed. For example, in cystic fibrosis the ∆F508 mutation has a frequency of 74.5%; evaluating for this
and the 15 other most "common" mutations gives a detection rate among North American whites of approximately 86%.1
Thus, direct detection still will not detect 100% of individuals with a cystic fibrosis allele. In Gaucher's disease, four mutations
account for nearly 96% of the Gaucher's disease mutations in the Jewish population.2
In cases where there is a family history of a particular disease and direct analysis of mutations fails to detect any documented
mutation, indirect analysis by linkage with polymorphic markers is usually a reliable alternative. Analysis of polymorphisms is
successful if the polymorphism employed is closely associated with the disease allele within a family or if it is specifically not
associated with a disease allele. Although such indirect detection techniques are useful, they also increase difficulty of
analysis owing to (I) the need for testing several (often many) family members, (2) nonpaternity, or (3) parental
polymorphisms uninformative at a particular locus.
One strategy for nearly 100% detection of any particular disorder is the use of indirect detection when direct detection fails.
In our cystic fibrosis example, after analysis of common mutations, linkage analysis of uncommon ("private") mutations-currently greater than 400--may be used if enough family members are available for study. Unfortunately, given the current
limits on available technology, assay for all documented mutations that cause cystic fibrosis is impractical, due to the cost and
labor involved. This is the basis for recommendations against general-population screening (i.e., in persons without affected
first- degree family members).
DNA Analysis Techniques
Some techniques commonly used in DNA analysis are described briefly below. For detailed information, consult the
appropriate references cited. An excellent source for all molecular genetics-related methods is the three-volume laboratory
text by Sambrook, Fritsch, and Maniatis.3
DNA isolation
One reason for the rapid application of molecular genetic techniques in DNA diagnostics has been the ease of obtaining DNA
from virtually any tissue or fluid, owing to the stable nature of DNA and the small quantity required for most DNA diagnosis.
DNA is present in all nucleated cells, and each cell contains DNA comprising the whole of an individual's genetic constitution.
DNA can be extracted from blood (leukocytes), amniotic fluid (amniocytes), chorionic villi samples (CYS), and all other tissue
types.
Table 1
DNA Content
Sample
One cell
Amount
~7 pg
One plucked hair
~0.3 µg (300 ng)
One shed hair
~0.1 ng
One drop blood
~1.5 µg
One drop semen
~10 µg
1 mL blood
~40 µg
1 mL amniotic fluid
~0.35 µg
1 mg chorionic villi
~1 µg
One T25 flask cultured cells
~30 µg
Human genome
3 billion bp (haploid)
1 µg genomic DNA
333,000 copies
10 ng
3,300 copies
6 pg
Single copy of genome
- 16 -
The extraction procedure involves the collection of cells from the fluid by centrifugation,. followed by disruption of the cellular
wall, then either digestion of all proteins by proteinase K or selective precipitation of proteins by salts. The DNA remains
soluble, and the DNA-containing solution is mixed with organic solvent in the presence of salt to precipitate the DNA. The DNA
is further purified in a few steps to yield pure DNA suitable for all types of manipulation.
Purified DNA, in the absence of DNAses (enzymes that selectively break down DNA), is stable and can be stored in solution
form at -20°C or -70°C. Repeated freeze-thaw cycles tend to break down high molecular genomic DNA into smaller fragments
of 50 to 100 kb in size.
Table 1 shows the approximate amount of DNA present in various tissue samples. A comparison is also given in terms of
number of copies of the human genome to expect when sample content is termed in weight units.
Hybridization analysis
Hybridization is the basis of nearly all DNA diagnostic methods. The seminal discovery by Watson and Crick in 19534,5
established the double-stranded, helical structure of DNA as well as the A- T and G-C base pairings on which the principles of
hybridization studies are founded. The specificity of complementary base pairing is such that a small oligonucleotide sequence
(probe) will identify and hybridize to its complementary sequence. This hybridization process occurs in less than 30 seconds
under appropriate conditions and is the principle behind the polymerase chain reaction (PCR), described in the next section.
Hybridization detection methods can be divided arbitrarily into two groups: gross detection and nucleotide level detection.
In gross detection, hybridization involves DNA probes greater than 200 bp in length (even though oligonucleotides can be
used). The resolution achieved may relate to the detection of DNA fragments on a gel (bands) representing a particular size,
such as in the restriction fragment length polymorphism (RFLP) used in assays for fragile X, or the absence of certain bands,
representing deletions, as is employed in Duchenne's muscular dystrophy (DMD) testing.
Gross detection is achieved using Southern blot analysis, a technique developed by E.M. Southern.6 It is a method based on
hybridization of DNA probes in solution to complementary sequences immobilized on membrane after electrophoresis
separation. Variations of this technique are Northern blot analysis, where RNA is bound to membranes after electrophoresis
separation; Western blot analysis, an immunology-based assay; and dot blot analysis, where DNA or RNA is bound to
membranes by direct application of the sample material without electrophoretic separation.
These blot analysis methods are powerful tools for identifying gene sequences. In the context of DNA diagnostics, the aim is
to locate a specific DNA fragment corresponding to a particular gene linked to a particular phenotype based on RFLP
hybridization, using probes that are specific for the particular gene fragment or markers that detect the RFLP employed. The
size of the probes used for Southern analysis is usually 200 bp or greater; however, probe size does not bear any direct effect
on the hybridization. In Southern hybridization, conditions are such that the technology cannot discriminate between single
base pair mismatches or small deletions--a limitation of the technique.
To detect a particular fragment of human DNA, the genomic DNA is cut into small pieces with restriction endonucleases,
bacterial enzymes that cleave DNA at specific sites based on its sequence specificity. The presence of these specific 6 to 8 bp
sequences in the human genome is random; thus, digestion of genomic DNA with any restriction enzyme leads to the
generation of millions of fragments of all sizes. No particular size is more enriched than another. To separate the differentsized fragments, the digested DNA is subjected to electrophoresis.
Visualization of genomic DNA after digestion with restriction endonucleases and electrophoretic separation reveals a lane with
a smear for each DNA sample. Transfer of the separated DNA to membranes capable of binding DNA is achieved either by
capillary blotting techniques or by vacuum-assisted methods that accelerate transfers. The transferred and immobilized DNA,
now affixed on the membrane, is a mirror image of that noted in the gel. Membrane hybridization with specifically labeled
probes leads to the specific annealing/binding of the probe only to its complementary band. The hybridization is detected by
autoradiography, in the case of radioactive probes, or by chemiluminescent method.
For nucleotide level detection, hybridization conditions that can discriminate between single base-pair mismatches are used.
This is usually performed with dot blot analysis, in which the probe size is 18 to 30 bp. These probes are called allele-specific
oligonucleotides (ASOs), also commonly called oligos. ASO hybridization conditions are achieved by either adjusting the
temperature of hybridization or by adjusting the salt concentration. ASO hybridization is rapidly gaining use in the detection
of all single base-pair mutations as well as small deletions. The advantage of dot blot analysis is the large number of samples
that may be processed simultaneously. The drawbacks of ASO hybridization are the use of radio-labeled probes and the
requirement of thorough optimization of hybridization conditions to discriminate between single base-pair mismatches. As
non-radioactive detection methods become more available, ASO techniques will rapidly become the principal method of
detection for most mutations. The use of ASO will be discussed in the section on cystic fibrosis.
- 17 -
Polymerase chain reaction
The polymerase chain reaction (PCR) is the single
most commonly used procedure in molecular
genetics. PCR was developed in 19857 and, due to
its ability to amplify specific regions of DNA several
million-fold,
has
since
become
the
major
contributing factor in the rapid pace of research in
molecular genetics.8,9
PCR is based on the enzymatic amplification of a
fragment of DNA that is flanked by two
complementary, short, oligonucleotide primers
whose sequence is known. These primers are
designed such that each corresponds to one of the
strands; the distance between these primers limits
the amplification fragment size. PCR is also based
on the property that in hybridization the primer,
under specific conditions, will bind (hybridize,
anneal) only to its cognate sequence in the DNA
sample. This process is termed annealing. The
annealing of the primer to target DNA is achieved by
denaturing the target DNA through exposure to high
temperature. Heat breaks the base-pair hydrogen
bonds and results in a separation of the two DNA
strands. Reducing the temperature favors base
pairing/hybridization, which is equally competed for
by the primers, present in excess.
Once the primers are correctly annealed, DNA
polymerase will elongate the primer by copying the
template sequence. The two primers are both
extended beyond the binding site, independent of
each other, resulting in the synthesis of variablesize fragments complementary to the template DNA.
The steps of denaturation, annealing, and elongation
constitute one cycle. In one cycle, a copy of the
target sequence is achieved. The second cycle of
amplification yields four copies, and so forth. The
amplification process occurs exponentially and can
result in the amplification of a target sequence
several million-fold. A routine PCR process consists
of 20 to 30 cycles, requiring 2 to 3 hours and using
an automated thermal cycling instrument. Under
proper conditions, a unique gene sequence from the
genome can be routinely amplified from 50 to 100
ng of target DNA. PCR has been used to amplify
sequences from one cell (approximately 7 pg DNA)
and a single hair (300 ng).
The use of Taq DNA polymerase was crucial to the rapid and exponential popularity of the technique.10,11 Initially, when PCR
was first introduced, a thermolabile DNA polymerase was used; unfortunately, the Warm denaturation temperatures (92°C to
94°C) inactivated the enzyme. Those active in the field may remember sitting in front of three water baths, adding aliquots of
DNA polymerase after each annealing cycle, and transferring the tubes to another water bath. The heat-stable Taq DNA
polymerase (an enzyme acquired from hot-spring bacteria) can withstand repetitive exposure to the warm temperatures
employed in the thermal cycling process with little loss of activity, thereby eliminating the need to add more enzyme after
each cycle. The use of Taq DNA polymerase also facilitated the introduction of automated DNA thermal cycler’s.
The rapid amplification of sequences of specific portions of the gene spanning the site of the mutation coupled with other
detection methods, has led to an expanded availability of DNA diagnostic methods in clinical medicine. For example, PCR can
also be use for mRNA amplification by first making a complimentary DNA copy that serves as the target DNA. DNA
complementary to mRNA is synthesized by the use of an enzyme called reverse transcriptase (RT); the process of using
mRNA to make cDNA followed by PCR is called RT-PCR. RT-PCR is valuable when the exact mutation is not known and
techniques for scanning mutations in short fragments is required, e.g., using single-strand conformation polymorphisms.3
- 18 -
Polymorphism Analysis
We earlier discussed the heterogeneity of the human genome. In this section we will examine how the application of these
DNA polymorphisms may be exploited for linkage analysis in the detection of a genetic disorder.
Two types of polymorphisms will be discussed: restriction fragment length polymorphisms (RFLPs), which have already been
introduced, and microsatellite repeat polymorphisms. Both these techniques have contributed immensely to the human gene
mapping effort. Although RFLP analysis has been in use for well over a decade, microsatellite repeat mapping is considerably
more recent.
Restriction fragment length polymorphism analysis
RFLP analysis is based on the observation that changes in the DNA create or abolish cleavage sites for restriction
endonucleases. Botstein et al12 first identified the existence of RFLPs and advocated their use as markers for linkage analysis.
Since then, extensive screening of the human genome has been accomplished by digestion with various enzymes, followed by
"probing" with specific cloned pieces of DNA. This methodology has established a data base, DNA markers, of polymorphic
sites of differing fragment sizes obtained following specific enzymatic cleavage.13,14 As noted earlier, a specific chromosomal
locus is termed polymorphic when there exist two or more alleles and each has a population frequency of greater than 1%.
For RFLP analysis, polymorphic alleles are selected that will provide a heterozygote frequency of at least 20%. RFLPs are
essential for the deduction of allelic inheritance, but are useful only when the parents are informative. The deduction of
inheritance of a particular phenotype using a specifically linked polymorphism is complex, most commonly established by
thorough and laborious effort in research-oriented laboratories. As noted earlier, RFLPs are most useful when methods of
direct detection of common mutations have failed (presumably due to the presence of an undocumented private mutation).
Indirect analysis made by deduction of allelic inheritance is demonstrated in Fig lB.
Microsatellite repeat polymorphisms
Microsatellite repeat polymorphism analysis is rapidly replacing the use of RFLPs for gene mapping. Microsatellite repeat
polymorphisms are small repeats, e.g., CA (dinucleotide repeat), that are interspersed approximately every 30 to 50 kb within
the human genome. Weber and May in 198915 demonstrated the existence of polymorphic microsatellites with alleles ranging
from 4 to 11 bp. The abundance and the highly polymorphic nature of microsatellite repeat polymorphisms make it ideal for
use as a mapping technique for allele inheritance patterns. Microsatellite repeat mapping technique involves PCR, usually in
the presence of a radioactive label, followed by electrophoretic separation on a sequencing gel. Microsatellite repeat mapping
is more versatile, powerful, and less time-consuming than RFLP.
Clinical Applications of Molecular Genetics
The following sections discuss the molecular genetics of several common inherited diseases. Also described are the methods
of molecular analysis used to determine the presence of genetic mutations, which are in turn used to assess an individual's
risk of developing the disease. The case presentation format illustrates the practical applications of molecular genetic
technology.
SICKLE CELL DISEASE
CASE. A couple presents for genetic counseling because the mother, who is 10 weeks pregnant, has a child from a previous
marriage affected with sickle cell disease. The father, a colonel in the army, is unavailable for testing due to his deployment
with NATO forces in Bosnia. Although the child has experienced few crises, all have necessitated hospitalization and repeated
blood transfusions. Recently, he demonstrated seropositivity to the human immunodeficiency virus (HIV).
Molecular genetics
The study of sickle cell anemia and other β-globin disorders holds special importance in the fields of medicine, physiology,
biochemistry, and molecular genetics. During the early 1940s, several groups independently started working on the genetics
and bio- chemistry of sickle cell anemia. In 1949, Neel16 showed that sickle cell anemia fits the pattern of a genetic disease
and is caused by the presence of two copies of a recessive allele, thus causing the disease in the homozygous state. Later the
same year, Pauling et al11 showed that hemoglobin in normal and sickle cell anemia patients differed by having different
electrophoretic mobility and, thus, different chemical properties. In 1957, Ingraml8 analyzed the α- and β-globin chains of
adult hemoglobin obtained from both normal subjects and patients with sickle cell anemia. In the latter group, he was unable
to demonstrate changes in the hemoglobin a chains; however, he found that each β chain had an amino acid substitution at
position 6, which resulted in a mutation from the normal ("wild-type") glutamic acid residue to valine. This was the first report
of a mutation being identified at the protein level as a cause of an inherited disease; Ingram's work also made apparent the
fact that subtle changes at the molecular level may lead to a clinically distinct disorder.
β -Globin was the first gene to be cloned and completely sequenced. The glutamic-acid-to-valine substitution has now been
further characterized, and the mutation at the DNA level has been established to be an A to T change in the second position
of the codon of glutamic acid, i.e., GAG to GTG. The resulting mutant globin chain is termed hemoglobin S (HbS). Hemoglobin
S is freely soluble when fully oxygenated. Under conditions of low oxygen tension, the red cells become grossly abnormal,
assuming a sickle shape that leads to aggregation and hemolysis. Homozygous HbS is a serious hemoglobinopathy found
almost exclusively in the black population. About 8% of African Americans are carriers--heterozygotes and about 0.2% are
affected--homozygotes. Heterozygotes (sickle cell trait) are clinically normal, although their red cells will sickle when
subjected to very low oxygen pressure in vitro.19
- 19 -
DNA testing for the sickle cell mutation is accomplished by specific amplification of the region spanning the mutation, using
polymerase chain reaction, followed by enzymatic cleavage of the amplified product. Sickle cell mutation eliminates a
restriction endonuclease site (Dde I), and electrophoretic resolution of the fragment pattern reveals the presence or absence
of the mutation. Clear diagnosis of normal, carrier, and homozygous DNA is readily achieved.
Molecular analysis
The example of sickle cell mutation detection illustrates how creation or abolition of a restriction endonuclease site may be
due to a mutation. All mutations, however, are not as easily determined; for example, some mutations may not be in a DNA
sequence region recognized by any known restriction endonuclease. In such cases, an artificial restriction site may be
introduced by mismatch primer construction, which can then discriminate between wild-type normal and mutant sequence.
This is discussed with examples under Gaucher's disease.
In sickle cell disease, the A to T base mutation abolishes a cleavage site for restriction endonuclease Dde I and Mst II7 To
detect the sickle cell mutation using the Dde I restriction enzyme, two specific oligonucleotide primers are used to amplify a
233 bp fragment within the β -globin gene by PCR. The A to T mutation and the Dde I site are in the middle of the fragment.
After PCR amplification, the amplified product is subjected to restriction endonuclease Dde I digestion; only the βA-globin
fragment is cut by Dde I to yield a 178 bp and a 55-bp fragment. Heterozygous individuals with both a βA - and a βS -allele
will yield fragment patterns in which half the molecules cleave to 178 +55 bp and half fail to cleave, leaving a 233 bp
fragment.
Homozygous affected individuals will have only βS -globin, and thus the PCR-amplified 233 bp fragments from both alleles will
have lost the Dde I site and will remain uncleaved. The PCR-amplified, Dde l-digested DNA is electrophoresed to resolve the
fragments. Visual inspection of the gel for DNA fragment pattern reveals the presence or absence of the mutation in question.
Clear diagnosis of normal, carrier, and homozygous DNA is achieved. Figure below shows the fragment pattern obtained for
sickle cell using the above detection method.
Alternate methods using allele-specific oligonucleotide and Southern blotting techniques with Mst 11- or Dde I-digested DNA
and hybridization with a β-globin gene probe have also been reported.7,20,21
Hb-A: …TCCTGAGGAG…
Hb-S: …TCCTGTGGAG…
Hb-C: …TCCTAAGGAG…
PCR Product Fragment Size 233 bp
Fragment Sizes After Dde I Digestion
A/A
A/S
S/S
178+55 bp
233+178+55 bp
233 bp
Typical Sickle cell genotype analysis of PCR product
digested with Dde I. Lane 1 is molecular weight markers.
Lane 2 is undigested PCR product. Lanes 3, 4 and 6 is
DNA with A/S geneotype. Lane 5 is A/A genotype DNA
and Lane 7 represents DNA with S/S genotype. Mutation
abolishes restriction site.
- 20 -
CYSTIC FIBROSIS
CASE. A couple presents to your office for genetic counseling because the mother and their two only children have cystic
fibrosis. They are hopeful about the possibility of having a child unaffected with this condition.
Molecular genetics
Cystic fibrosis is the most common recessive disorder affecting the white population, with a heterozygote frequency of
approximately 1 in 25.19 Recently, the gene responsible for cystic fibrosis has been identified: the cystic fibrosis
transmembrane conductance regulator (CFTR). Rapid characterization of the mutations and their frequency has made possible
DNA testing for CF in at-risk families.1
A single mutation, termed ∆F508, accounts for nearly 75% of CF mutations in the whites of northern European descent (Table
2). This particular mutation is due to an in-frame deletion of three bases, resulting in the deletion of amino acid phenylalanine
(F) at position 508.1 Most of the other mutations are single base changes resulting in amino acid substitution. Table 3 lists
some of the mutations. The CFTR gene has been thoroughly characterized, and the position of most mutations in the gene in
relation to protein structure/function have been established; details are beyond the scope of this discussion.
Analysis of five of the most common mutations (see Table 3) accounts for about 85% in the white population.1 The frequency
of ∆F508 mutation in Ashkenazic Jews is approximately 23%, whereas Wl282X mutation in exon 20 represents 60% of the
mutations; Wl282X mutation leads to a truncated protein. The five mutations listed in the table for the Ashkenazic population
account for nearly 96% to 98%.22
Molecular analysis
Development of new mutation analysis methods for cystic fibrosis has been very active, primarily because of the high carrier
rate and also due to carrier screening prospects. The traditional sample for DNA is blood or amniotic fluid/culture for prenatal
diagnosis. Recently, methods have been reported for DNA prepared from buccal brushing/swab, followed by multiplex PCR
amplification and ASO hybridization.23,24 In this section we will discuss PCR/restriction and ASO methods.
Nearly all the mutations analyzed in the evaluation of mutant CF alleles either
create or abolish a restriction endonuclease site, with the exception of the ∆F508
mutation--a 3 bp deletion. To analyze the mutations, specific corresponding
fragments of the CFTR gene are amplified by PCR. For the ∆F508 mutation, the
product after PCR will be 3 bp shorter than the normal (wild-type) sequence.
Electrophoresis of PCR-amplified fragments from homozygous normal,
heterozygous, and homozygous ∆F508 will reveal fragment patterns in which the
homozygous normal and the homozygous affected will manifest only one specific
band of the amplified product.
In heterozygotes (carrier) individuals there will be two fragments, one of which
is 3 bp smaller than the other. The smaller fragment represents the ∆FS08deleted allele; the larger band represents the normal allele.
In affected individuals-∆FSO8 homozygotes-- there will be a single band with the
same mobility as the smaller band in the heterozygous sample. This single band
actually represents two fragments of the same size traveling together, amplified
from the two ∆FSO8-deleted allele segments. Analysis of the ∆FS08 mutation is
straightforward, involving PCR specific for exon 10 ∆FSO8 mutation-spanning
region, followed by polyacrylamide electrophoresis. The interpretation of the gel
patterns is clear and reliable.
For other mutations of the CFTR gene, the strategy is similar to that described
above for sickle cell disease. Following specific PCR amplification, the PCR
product is subjected to restriction endonuclease digestion to monitor cleavage of
the DNA. Table 4 lists the different enzymes used and the expected fragmentsize products on electrophoresis.
Table 2
Frequency of CF Carrier
White Americans of European Descent
1 in 25
Ashkenazic Jews
1 in 29
Hispanic Americans
1 in 45
African Americans
1 in 60
Asian Americans
1 in 150
- 21 -
Table 3
CF Mutation Analysis
White (non-Jewish)
Mutation
Ashkenazic Jews
W1282X (60%, exon 20)
∆F508 (75.8%, exon 10)
G542X (2.7%, exon 11)
∆F508 (23%, exon 10)
G551D (3.2%, exon 11)
G542X (4%, exon 11)
R553X (1.4%, exon 11)
N1303K (4%, exon 21)
N1303K (1.4%, exon 21)
3849-10 kb C-T (4%)
Table 4
Cystic Fibrosis PCR/Restriction-Based DNA Analysis: Fragment Sizes
PCR
product
Normal
Homozygous
Restriction
(bp)
(bp)
(bp)
enzyme
∆F508
Carrier
(bp)
79/79
76/76
79/76
G542X
Bst NI
114
90+24
114
114+90+24
G551D/R553X
Hind II
114
55+59
114
114+55+59
N1303K
Bst NI
60
40+20
60
60+40+20
W1282X
Mnl I
473
178+172+123
301+172
301+178+172+123
CT3849
Hph I
437
349+88
222+127+88
349+222+127+88
Allele-specific oligonucleotide analysis
Another powerful technique used for mutation detection is allele-specific oligonucleotide (ASO) hybridization.25 In this
technique, small oligonucleotides in the size range of 18 to 26 bp are synthesized corresponding to the normal and mutant
DNA sequence. The normal and mutant ASOs differ by only 1 bp. These oligonucleotides are usually end-labeled with 32P and
used for very specific hybridization and washing conditions. Under appropriate conditions, the normal ASO will hybridize only
to the wild-type sequence, and mutant ASO, only to the mutant sequence. The labeled ASOs are used as probes to hybridize
with PCR-amplified DNA fragments corresponding to each mutation-spanning region. The DNA fragments are applied to a
DNA-binding membrane in duplicate as dots or slots, using a special apparatus called a dot/slot blot apparatus. This
apparatus has exact-size holes to make all applications uniform in size. The duplicate membranes are each hybridized to
normal and mutant ASO; the result after hybridization is tabulated by scoring for absence or presence of hybridization with
each ASO. Hybridization with only the normal ASO signifies the presence of normal sequence for that mutation; positive signal
hybridization with both normal and mutant ASO indicates a heterozygous sample; and positive hybridization only with the
mutant ASO signifies homozygous abnormal status. The use of ASO hybridization requires exact optimization of the melting
temperature and washing conditions to discriminate between single base-pair hybridization. If properly performed, the ASO
hybridization technique holds the most promise in terms of speed, ability to handle a large number of samples, and persample cost.
GAUCHER'S DISEASE
CASE. A teenager and her mother present to your clinic for counseling and therapy for osteonecrosis of the femoral head,
noted on an admission radiograph during her recent hospitalization for anemia, thrombo- cytopenia, and hypersplenism. Her
clinical symptoms predominantly include bone pain, which requires bed rest and absence from school. She has had two
episodes of pneumonia, also requiring hospitalization.
Molecular genetics
Gaucher's disease is the most common glycolipid storage disease; it is due to a deficiency of glucocerebrosidase. Gaucher's
disease has, in the Jewish population, an estimated heterozygote frequency of approximately 9%.2 The gene has been cloned
and characterized. A pseudogene is also present, which complicates DNA analysis (discussed later).
The four mutations listed in Table 5 account for approximately 96% of the Gaucher's disease mutations in the Jewish
population.26 Mutation 1226G is the most common cause of Gaucher's disease in Jewish patients and is associated with mild,
late-onset clinical phenotype. Only about one third of patients with the 1226G/1226G mutations have Gaucher's disease.
Patients who are compound heterozygotes for mutations 1226G and 84GG have a more severe clinical disorder than those
who are homozygous for the 1226G mutation. The median age at first onset of symptoms in patients with Gaucher's disease
having the 1226G/1226G or the 1226G/84GG mutation is 30.5 years and 6 years, respectively. There has been no report of
patients homozygous for the 84GG mutation, indicating that it would be a perinatal lethal condition. Mutation 1448C is
associated with a more severe phenotype compared with the 1226G mutation. Patients with 1448C/I448C genotype generally
manifest severe neuronopathic Gaucher's disease; patients with a homozygous IVS2 mutation are also severely affected.
- 22 -
Beutler and colleagues26 identified a total mutation frequency of approximately 0.031 in the Ashkenazi Jewish population. The
frequency of the 1226G mutation is about 0.028, and that of the 84GG mutation is 0.0028. Thus, the frequency of alleles
other than 1226G, 84GG, and 1448C would be 3.3% of the total, or 1 X 10-3. A Jewish couple who is negative for the 1226G,
84GG, and 1448C will therefore have only an approximately 1: 1,000,000 risk of having a child with Gaucher's disease. On
the other hand, if one partner has one of the three "common" mutations and the other none of these three, then the risk will
be increased to approximately 1: 1000.
Molecular analysis
DNA analysis for Gaucher's disease mutations is accomplished by specific PCR amplification, mismatched PCR (discussed
below), followed by restriction endonuclease digestion and/or ASO hybridization. We previously discussed PCR followed by
restriction endonuclease digestion and ASO. Here we will discuss a new method of mutation detection, mismatched PCR.
Not all mutations result in the gain or loss of a restriction site. Such mutations therefore cannot be analyzed by
PCR/restriction endonuclease method. ASO requires the use of radioactivity and thorough optimization. The mismatched PCR
method was introduced by Beutler et al27 to overcome these difficulties. In this method, one of the primers for PCR is
constructed in a way that the 3' end of the DNA strand adjacent to the site of the mutation and the internal sequence of the
primer is altered so that a restriction endonuclease site will either be gained or lost once the PCR product is amplified.
Gaucher's disease mutation 1226G (also known as N370S);in this mutation, an A is changed to a G at position 1226, leading
to the substitution of the amino acid serine for asparagine. This mutation does not create or abolish a site for any
known/commercially available restriction endonuclease. One of the PCR primers is constructed with a mismatch, as shown in
Fig 3B. Primers with internal mismatches will hybridize to target sequences at optimized conditions, and elongation of this
primer with a normal template wil1 result in the addition of an A residue; in the mutant template, a G residue will be added.
The use of the mismatched primer in concert with a 1226G mutant template creates a new Xho I restriction endonuclease site
(Table 6). Digestion of PCR products from normal and 1226G mutant templates is fol1owed by electrophoretic separation. The
result wil1 be two fragments for the mutant product (it will be cleaved), whereas the normal product remains uncleaved,
resulting in visualization of a single, higher molecular weight fragment. This technique is reliable, and it is performed in a
fashion very similar to PCR, fol1owed by restriction endonuclease digestion. This mismatched PCR method may also be used
for the 84GG Gaucher mutation.27
Table 5
Gaucher’s Disease Mutation Analysis
Mutation
Mutation
Frequency
1226G (N370S)
75% (25% non-Jewish)
84GG
13%
1448C (L444P)
5% (40% non-Jewish)
IVS2+1
3%
Table 6
Gaucher’s Disease PCR/Restriction-Based DNA Analysis: Fragment Sizes
PCR
product
Normal
Homozygous
Restriction
(bp)
(bp)
(bp)
enzyme
1226G
Xho I
84GG
105
Carrier
(bp)
105
89+16
105+89+16
75
57+18
75+57+18
Bsa BI
75
1448C (1st)
Pst I
677
1448C (2nd)
Nci I
102
102
57+45
102+57+45
IVS2+1
Hph I
357
141+117+99
240+117
240+141+117+99
- 23 -
FRAGILE X SYNDROME
CASE. A couple presents for genetic counseling because of a family history of mental retardation. The affected girls are
described as "slow." Some of the boys in the family are "slower than the girls," some were hyperactive (autistic) as children,
and some of the "slow" men are reported to have large, flat ears.
Molecular genetics
Identification of the FMRI gene and establishment of its expansion as the cause of fragile X syndrome led to the classification
of a new class of mutation.28,29 Fragile X syndrome is associated with amplification of a triple-repeat CGG in the FMRI gene,
the severity of the disease being related to the size of the amplification. The genetics of fragile X syndrome are complicated,
but may be better understood if one groups the expanding mutations into two broad categories: premutations and full
mutations.
Premutations are found in normal transmitting males (NTMs), individuals who transmit the mutation to grandsons but are
unaffected themselves, and carrier females. Premutations involve the amplification of the CGG triple repeat to approximately
70 to 200 copies. Numbers of repeats in this range are considered stable. Normal individuals possess less than approximately
50 copies of the triple repeat, and individuals with full mutations of the fragile X syndrome have 200 to 1000 copies of the
CGG triple repeat. The size of expansion is heterogeneous within an individual and thus signifies somatic instability of the
mutant allele.
The mode of transmission of the fragile X mutation is unusual and makes both understanding the genetics and counseling
patients difficult. Phenotypically normal males possessing a premutation are normal transmitting males, as noted above, and
father-to-daughter transmission is not accompanied by expansion of the triple-repeat mutation. Thus, daughters of NTMs are
never found to be affected.
The change from premutation to full mutation occurs only in females and may be, through them, transmitted to their
offspring. The risk of expansion of a premutation to a full mutation varies, depending on the size of the premutation. This new
class of triple-repeat expansion mutation has now been documented in several other genetic disorders, including Huntington's
disease and myotonic dystrophy.30
Molecular analysis
The detection of DNA amplification/expansion regions may be accomplished by PCR and Southern hybridization. The following
methods can be used for all disorders involving a variable increase in the size of a specific region of DNA. Analysis for the
direct detection of fragile X mutation is based on the enzymatic amplification of a fragment containing the CGG repeat
sequence of the FMRI gene, and it is most commonly performed with a modification of the amplification protocol published by
Fu et al.31
This protocol detects the fragile X mutation by the size of the amplified product. An increase in size is correlated with the
corresponding number of CGG repeats, following which a risk is calculated. The most common allele in the unaffected, normal
population consists of 29 repeats, the range varying from 6 to 54 repeats. Premutations in fragile X families showing no
phenotypic effect range in size from 52 to greater than 200 repeats; however, all alleles with greater than 52 repeats are
meiotically unstable.
PCR-based methods are fundamentally similar to those presented earlier in this article. The two primers are constructed such
that they span the region of triple-repeat expansion; however, in the case of fragile X specifically, the nature of the mutation
poses problems using normal PCR conditions: the CGG repeat may be hundreds to thousands of bases in length. All DNA
polymerases, including Taq DNA polymerase, do not efficiently copy prolonged stretches of G residues. Therefore, in fragile X
studies, an analog of G (7-deaza GTP) functions more efficiently and is therefore incorporated into the PCR reaction to achieve
optimal amplification. Unfortunately, the use of 7 -deaza GTP mixtures precludes the staining of gels with ethidium bromide,
minimizing visualization (7-deaza GTP containing DNA does not stain well). The poor staining is resolved by using
radioactively labeled nucleotide, followed by autoradiography.
Fragile X PCRProber™ Results
Fragile X PCR blot.
Lane 1 pre-mutation female;
30/60 CGG repeats.
Non-radioactive detection,
~2 hr. exposure.
Fragile X GeneProber™ Results
Fragile X southern blot.
Lane 1 affected female.
Lanes 2, 3 & 5 are normal
males.
Lane 4 normal female.
Non-radioactive detection,
~2hr. exposure
- 24 -
Fragile X PCR still does not give accurate results for full mutations due to the presence of massive CGG triplet expansions,
because, as noted above, PCR does not amplify very large fragments containing repetitious G residues efficiently. Although
normal and premutation PCR amplifications are reliable, all amplifications performed in our laboratory on subjects who may
possess full mutations are run by both PCR and Southern hybridization techniques. The PCR results are obtained in 2 days;
Southern blot hybridization studies require more time for complete results.
Southern blot analysis for fragile X mutation detection involves the cleavage of DNA with enzymes Eco RI and Eag I and is
based on the protocol published by Rousseau et al.32 The Rousseau method identifies the size of CGG repeat region and
accomplishes this by hybridizing probe GLFX1 to DNA that has been previously double-digested with restriction enzymes Eco
RI and Eag I. The sample is then blotted onto a membrane.
In normal females two fragments are seen, a 2.8-kb fragment corresponding to the active X and a 5.2-kb fragment
corresponding to the methylated, inactive X chromosome. Normal males exhibit only the 2.8- kb banding pattern. Affected
males will have an amplified CGG repeat region with methylation, thus giving rise to fragments larger than the normal 5.2 kb.
Premutations in both males and females will be seen as 2.9- to 3.3-kb fragments (normal, 2.8 kb) derived from the active X
chromosome. Premutations in females derived from the inactive X will manifest fragments from 5.3 to 5.7 kb in length.
Mosaicism is characterized by fragments appearing as a mixture of full mutation (methylated, larger than 5.7 kb) and
unmethylated premutation (2.9 to 3.3 kb) fragments.
DUCHENNE'S MUSCULAR DYSTROPHY
CASE. The final case in your morning clinic is a 13-year-old boy who has been experiencing progressive muscular weakness
and pseudohypertrophy of the calf muscles. His mother is concerned about recurrence risks for this condition in her future
pregnancies.
Molecular genetics
The dystrophin gene is the largest gene thus far identified, being approximately 2300 kb (2.3 million base pairs) in size.
Almost 50% of all patients with Duchenne's muscular dystrophy (DMD) have deletions in the dystrophin gene, likely due to its
unusually large size. In addition, the dystrophin gene has an exceedingly great number of new mutations, also attributed to
size. Finally, one third of these mutations are new, while those remaining are inherited through heterozygous females; DMD is
lethal in males.
Becker type muscular dystrophy (BMD) is an allelic form of DMD and is due either to mutations in the dystrophin gene, which
do not cause total loss of protein function, or to deletions that do not cause change of reading frame. Frame-shift mutations
are small deletions that cause a shift in the reading frame, leading to production of a truncated gene product. In-frame
deletion on the other hand, result in the removal of a portion of the amino acid sequence, thereby allowing some retention of
functional activity.19
Molecular Analysis
The dystrophin gene has more than 70 exons. As a result, the usual approach to detecting base-pair level mutation is not
practical. Therefore, with DMD, the initial goal is screening for deletions.
Studies of regions where there is a high incidence of deletions have shown that 9 to 12 exons account for 80% to 90% of all
dystrophin gene deletions. The detection method uses PCR amplification of these exons, a procedure termed multiplex PCR.33
In multiplex PCR, analysis of several pairs of primers, added in the same tube concurrently, allows independent gene
sequence amplification. In DMD studies, 9 to 12 primer pairs are employed simultaneously. In a normal DNA template, PCR
analysis with all primer pairs should yield a specific-size product; therefore, multiplex PCR allows analysis of each of the 9 to
12 exons by using primer pairs specific to each likely exon deletion in the dystrophin gene. As with traditional PCR,
electrophoretic separation and visualization reveal the presence or absence of any given deletion being evaluated. Obviously,
the design of the multiplex primer set is crucial for PCR to give reliable results. A few important considerations are: (1) The
primers should not have extensive complementary region; (2) the melting temperature of all the primers should be in the
same range, so that a specific annealing temperature could be selected; and (3) the specific PCR products are distinguishable
in size.
Individuals with a family history of DMD but lacking a specific, detectable deletion are tested by haplo-typing and linkage
analysis, using a predefined set of both intragenic and flanking DNA markers. Linkage analysis requires the evaluation of the
entire family to satisfactorily predict the inheritance pattern of the disease.
- 25 -
REFERENCES
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31.
32.
33.
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Bcudcr E. Gaucher disease: New molecular approaches 10 diagnosis and treatnent. Science 1992;256:794-799.
Sambrook J, Fritsch EF, Maniatis T. Molecu/ar Cloning A Laboratory Manual 2nd. ed.Cold Spring Harbor, NY: Cold Spring Harbor Laboratory
Press; 1989.
Watson JD, Crick FHC. Molecular structure of nucleic acids A structure for deoxyribose nucleic acid. Nature 1953;171:737-738.
Watson JD, Crick FHC. Genetical implications of the structure of deoxyribonucleic acid. Nature 1953;171:964-967.
Southern EM., Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975;98:503-5 17.
Saiki RK, Scharf SJ, Faloona F, Mullis KB, et aI. Enzymatic amplification of beta-globin sequences and restriction site analysis for diagnosis
of sickle cell anemia. Science 1985;230:1350-1354.
Mullis KB The unusual origin of the polymerase chain reaction. Sci Am 1990;262:56-65.
Innis MA, Gelfand DH, Sninsky JJ.,White TJ. eds PCR Protocols: A Guide to Methods and Applications. New York, NY: Academic Press;
1990.
Saiki RK. Gelfand DH. Stoffel S. ScharfSJ. et al Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase
Science 1988;239487-491.
Chien A, Edgar DB, Trela JM Deoxyribonucleic acid polymerase from the extreme thermophile Thermus, aquaticus. J Bacterio 1976; 1271
550-1 557.
Bolotein D. White R, Skolnick M, Davis R Construction of a genetic linkage map in man using restriction fragment length polymorphism Am
J Hum G~n~11980;32314- 331.
White R, Lalouel J-M Chromosomal mapping with DNA markers Sci Am 1988;25840-48.
Weisscnbach J, Gyapay G, Dib C, Vignal A, et al A second generation linkage map of the human genome Nature 1992;359:794-801.
Weber 1M, May PE Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction Am J Hum
Genet 1989;44388-396.
Neel JY The inheritance of sickle cell anemia Science 1949; II 064-66.
Pauling L, Hano HA, Singer SJ, Wells IC Sickle cell anemia, a molecular disease. Science 1949; II 0543-548.
Ingram VM Gene mutations in human haemoglobins The chemical difference between normal and sickle cell hemoglobin Nature 1957;
180326-328.
Thompson MW, Mcinnes RR, Willard IfF Genetics in Medicine Philadelphia, Pa WB Saunder.; 1991.
Wu DY, UgolZoli L, Bijay PK, Wallace RB Allele-specific enzymatic amplification of β-globin genomic DNA for diagnosis of sickle cell anemia
Proc Noll Acad Sci 1989;86:2757-2760.
Conner BJ, Reyes AA. Morin C. ltakura M. et al Detection of sickle celllJ'-globin allele by hybridization with synthetic oligonucleotides Proc
Noll Acad Sci 1983;80:278-282.
Abeliovich D. Lavoo IP. Lorer I, Cohen T, et al Screening for five mutations detects 97% of cystic fibrosis (CF) chromosomes and ~cts a
carrier frequency of 129 in the Jewish Ashkenazi population. Am J Hum Genel 1992;51951-956.
Bombard AT, Bartholomew DW, Neeno T. Rigdoo DT Value of mouth-washings as
a sow.:e for beterozygote DNA analysis by PCR: Comparison to peripheral blood Am JObsret Gyneco/l99I;I64(I):351.
Ricbards B. Skoletsky J, Shuber A, Balfour R, et al. Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal
brushes/swabs. Hum Mol Genet 1993;2:159-163.
25.. Shuber AP, Skoletsky J, Stem R, Handelin B Efficient 12-mutation testing in the CFTR gene: A general model for complex mutation
analysis Hum Mol G~net 1993;2:153-158.
Beuder E, Nguyen NJ, Henneberger MW. Smolec 1M. et al. Gaucber disease: Gene frequencies in the Ashkenazi Jewish populatioo. Am J
Hum Genet 1993;52:85-88.
Beutler E, Gelbart T. West C. The facile detectioo of the nt 1226 mutation of gluco- cerebrosidase by "mismatched" PCR. Clio ChimAcla
1990;194:161-166.
Yu S, Pritcbard M, K=ner E, et oJ. Fragile X genotype dIaracterized by an unstable regiooofDNA.Scimce 1991;252:1179-1181.
K=ner EJ, Pritcbard M, Lynch M, et al. Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG).. Science
1991;2521711-1714
Caskey CT, Pizzuti A, Fu V-H, Fenwick R. et al. Triple repeat mutations in human disease. Science 1992;256:784-789.
31.. Fu Y-H, KubJ A, Pizzuti A, et al. Variation of the COG repeat at the fragile X site results in genetic instability Resolution of the Sherman
paraOOx. C~I/ 1991;67:1047- 1058
Rousseau F. Heitz D, Biancalana V, et al. Di=t diagnosis by DNA analysis of the fragile X syndrome ofmentalretaldation. N £ngl J Med
1991;325:1673-1681.
Cbamberlain JS. Gibbs RA, RWer JE, Nguyen PN. et al Deletioo screening of the DuchelUle muscular dystrophy locus via multiple DNA
amplificatioo. Nuc/~ic Acids RcsI988;16:11141-11156.
- 26 -
Gene Detection Systems
- 27 -
- 28 -
Gene Link Gene Detection Systems
Introduction
Our present understanding of the molecular basis of genetic disorders is principally due to the recent clinical advances in
recombinant DNA techniques. The revolution in recombinant DNA technology has improved our understanding of simple
mutations as causes of disease. The molecular basis of genetic disorders is as varied as clinical genetics itself. The molecular
etiology of disorders may be fundamentally straightforward, such as in sickle cell disease, which is the best understood and
the first disease whose mutation was established at the DNA level. On the other hand, molecular genetics has delineated a
whole new class of disease where anticipation is involved; that is, the phenomenon of apparently increasing disease severity
in successive generations. Addressing the etiology and molecular diagnosis of the more complex disorders that involve
anticipation (e.g., Fragile X Syndrome, Myotonic Dystrophy, and Huntington’s Disease) is often challenging. The field of
molecular genetics has been the motivating factor for approaches to clinical molecular genetics.
The choice of using methods for mutation analysis is restricted by our knowledge of the existence of a mutation and its type
and in parallel to the development of safe and sensitive new detection methods. Gene Link’s primary focus is in the
development of facile non-radioactive detection methods. This product profile of Gene Link’s current gene detection product
line spotlights chemiluminescent detection based products as well as conventional radioactive based methods. The gene
detection systems can be divided into five broad groups based on detection methods:
- 29 -
Genemer™ Products: It comprises of primer pair for *PCR amplification of the fragment of interest and visualization of the
product by gel electrophoresis and ethidium bromide staining.
PCRProber™ Products: It comprises of primer pair for PCR amplification of the fragment of interest followed by Southern
blot and chemiluminescent detection using an alkaline phosphatase oligonucleotide probe.
GeneProber™ Products: A specific gene fragment probe for Southern blot based hybridization of genomic DNA. The
GeneProber is available unlabeled for radioactive based methods and labeled with digoxigenin for chemiluminescent detection.
GScan™ Products: It comprises of primer pair for PCR amplification of the fluorescently labeled fragment of interest for
analysis using fluorescent genetic analyzers.
Genemer™ Control DNA: These are cloned fragment of the mutation region of a particular gene for use with gene or
mutation specific Genemer™ products. These control DNAs are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA.
Non-radioactive detection methods based on hapten labeled, fluorescent labeled or directly labeled
with alkaline phosphatase.
- 30 -
Fragile X Syndrome
- 31 -
- 32 -
Fragile X Syndrome
Background
Fragile X syndrome is the most common form of inherited mental retardation. It affects approximately 1 in 1200 males and 1
in 2500 females. As suggested by the name, it is associated with a fragile site under specific cytogenetic laboratory conditions
at position Xq27.3 (1).
The inheritance pattern of fragile X puzzled geneticists, as it did not follow a clear X linked pattern. Approximately 20% of
males who are carriers based on pedigree analysis do not manifest any clinical symptoms and are thus termed as Normal
Transmitting Males (NTM), mental retardation is rare among the daughters of male carriers. Approximately 35% of female
carriers have some mental impairment. Based on the above it has been proposed that there are two states of the mutation,
one mutation range in which there is no clinical expression (premutation), which could change to the disease causing state
predominantly when transmitted by a female (full mutation)(2).
The fragile X syndrome gene (FMR-1, fragile X mental retardation ) was cloned in 1991 simultaneously by three groups (3-6).
Soon the peculiar genetic mode of transmission was established and a new class of mutation came into existenceTrinucleotide repeats amplification. This explained the clinical state of ‘premutation’ and ‘full mutation’ as well as
‘anticipation’. The fragile X syndrome is caused by the amplification of CGG repeats, which is located in the 5’ region of the
cDNA. The most common allele in the normal population consists of 29 repeats, the range varying from 6 to 54 repeats.
Premutations in fragile X families showing no phenotypic effect range in size from 52 to over 200 repeats. All alleles with
greater than 52 repeats are meiotically unstable with a mutation frequency of one. In general repeats up to 45 are considered
normal, repeats above 50 to 200 are considered as premutation and above 200 as full mutation (3-7). The range between 4055 is considered even by most experienced clinical geneticists and molecular geneticists very difficult to interpret and is
considered as a ‘gray zone’ with interpretations made on a case-by-case basis (8).
- 33 -
Trinucleotide Repeats in Human Genetic Disease
Repeata
Normal Lengthb
Fragile XA (FRAXA)
Fragile XE (FRAXE)
Fragile XF(FRAXF)
FRA16A
Jacobsen Syndrome (FRA11B)
Kennedy Syndrome (SMBA)
Myotonic Dstrophy (DM)
(CGG)n
(CCG)n
(CGG)n
(CCG)n
(CGC)n
(CAG)n
(CTG)n
6-52
4-39
7-40
16-49
11
14-32
5-37
Intermediate Length
(Premulation)a,b
59-230
? (31-61)
?
?
80
?
50-80
Huntington disease (HD)
Spinocerebellar ataxia 1 (SCA1)
Spinocerebellar ataxia 2 (SCA2)
Spinocerebellar ataxia 3 (SCA3)
/Machado Joseph disease (MJD)
Spinocerebellar ataxia 6 (SCA6)
Spinocerebellar ataxia 7 (SCA7)
Haw River syndrome (HRS; also
DRPLA)
Friedreich ataxia (FRDA)
(CAG)n
(CAG)n
(CAG)n
(CAG)n
10-34
6-39
14-31
13-44
36-39
None Reported
None Reported
None Reported
230-2,000
200-900
306-1,008
1,000-1,900
100-1,000
40-55
80-1,000; congenital,
2,000-3,000
40-121
40-81
34-59
60-84
(CAG)n
(CAG)n
(CAG)n
4-18
7-17
7-25
None Reported
28-35
?
21-28
38-130
49-75
(GAA)n
6-29
? (>34-40)
200-900
Disease
Full Disease Lengthb
Typically, repeats tracts contain sequence interruptions. See Pearson and Sinden (1998a) for a discussion of the sequence interruptions.
b No. of triplet repeats.
c A question mark (?) indicates potential mutagenic intermediate length, and an ellipsis (…) indicates none. Not all disease are associated with a
permutation length repeats tract or permutation disease condition.
a
CGG TRINUCLEOTIDE REPEATS PERCENTAGE AND FRAGMENT SIZE
CGG
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
bp
3
6
9
12
15
18
21
24
27
30
33
36
39
42
45
48
51
54
57
60
63
66
69
72
75
78
81
84
87
90
Size
223
226
229
232
235
238
241
244
247
250
253
256
259
262
265
268
271
274
277
280
283
286
289
292
295
298
301
304
307
310
%
0.18
0.18
0.35
6.32
0.18
0.88
6.14
2.63
0.88
1.4
0.88
2.28
18.78
38.77
CGG
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
bp
93
96
99
102
105
108
111
114
117
120
123
126
129
132
135
138
141
144
147
150
153
156
159
162
165
168
171
174
177
180
Size
313
316
319
322
325
328
331
334
337
340
343
346
349
352
355
358
361
364
367
370
373
376
379
382
385
388
391
394
397
400
%
7.02
3.51
1.23
0.53
0.7
1.05
0.35
0.53
1.23
1.23
0.35
0.7
0.7
0.18
0.18
0.18
0.18
0.35
- 34 -
Molecular Analysis
Fragile X genotyping can be done by direct PCR amplification of the CGG trinucleotide repeats region or by southern analysis.
In most cases both methods are used to complement the results. Full mutations usually cannot be identified by PCR by most
investigators and southern analysis is the preferred method to distinguish full mutations. The FMR-1 gene region containing
the CGG trinucleotide repeats is flanked by Eco RI sites; and an Eag I and Nru I site in the CpG region. Full mutation has been
shown to methylate the active gene too and thus it prevents NruI and Eag I restriction of DNA. Hybridization of southern blots
of Eco RI and Nru I or Eag I double digested DNA clearly can distinguish between normal, premutation and full mutation
genotypes.
The size of the CGG repeats can be determined by PCR analysis and sizing preferably on a sequencing gel. The PCR products
can be labeled fluorescently by using fluorescently labeled primers or with 35S or 32P followed by autoradiography. Another
attractive alternative is to run a cold PCR reaction followed by blotting and hybridization with an alkaline phosphatase
conjugated probe for non-radioactive detection.
The detection of amplification/expansion of a region of DNA sequence can be detected by PCR and Southern, these methods
can be used for all disorders involving increase in size of a region of DNA. DNA analysis for direct detection of fragile X
mutation is based on enzymatic amplification of a fragment containing the CGG repeat sequence of the FMR-1 gene. This test
detects the fragile X mutation by the size of the amplified product; an increase in size is correlated with the corresponding
number of CGG repeats and a risk factor calculated. The most common allele in the normal population consists of 30 repeats,
the range varying from 6 to 54 repeats. Premutations in fragile X families showing no phenotypic effect range in size from 52
to over 200 repeats. All alleles with greater than 52 repeats are meiotically unstable with a mutation frequency of one.
PCR based methods are fundamentally similar. The two primers are constructed such that they span the region of
trinucleotide repeat expansion. In the case of Fragile X specifically, the nature of the mutation poses problems using normal
PCR conditions. In Fragile X, the repeat is of CGG which can be hundreds to thousands bases long. All DNA polymerases,
including Taq DNA polymerase do not copy long stretches of G residues efficiently. An analog of G called 7-deaza GTP
functions better and is partially replaced in the PCR reaction to achieve amplification. The use of 7 deaza GTP instead of G
precludes the staining of gels with ethiduim bromide for visualization as 7 deaza GTP containing DNA does not stain well. This
has been clasically resolved by using radioactively labeled nucleotide followed by autoradiography. Fragile X PCR still does not
give accurate results for full mutations due to the inherent massive expansion and the inability of PCR to amplify very large
fragments efficiently. All normal and premutation PCR amplification is reliable, but still is coupled with a Southern blot
analysis. In our laboratory PCR is performed in addition to Southern blot analysis. The PCR results are obtained in 2 days
followed by Southern blot results. All results from PCR are verifiable by Southern except full mutations which are not reliable
with PCR.
Southern blot analysis for Fragile X mutation detection involves the cleavage of DNA with enzyme Eco R I and Nru I or Eag I.
This method detects the size of CGG repeats region by hybridization of probe GLFX1 or GLFXDig1 GeneProber™ to DNA that
has been double digested with restriction enzymes Eco RI and Nru I or Eag I and blotted onto a membrane. In normal
females two fragments are seen, a 2.8kb corresponding to the active X and a 5.2kb fragment corresponding to the
methylated inactive X chromosome. Normal males exhibit only the 2.8kb banding pattern. Affected males will have an
amplified CGG repeats region with methylation thus giving rise to fragments larger than the normal 5.7kb. Premutations in
males and females will be seen as fragments from 2.9-3.3kb (normal 2.8kb) derived from the X chromosome. Premutations in
females derived from the inactive X will give fragments from 5.3-5.7kb. Mosaicism is characterized by fragments appearing as
a mixture of full mutation (methylated, larger than 5.7kb) and unmethylated premutation (2.9-3.3kb).
Gene Link offers safe and reliable chemiluminescent detection methods as an alternate to radioactive based detection
methods. PCR-Prober™, GScan™and GeneProber™ line of products replaces radioactive based methods.
Detection Methods
The choice of using methods for mutation analysis is restricted by our knowledge of the existence of a mutation and its type
and in parallel to the development of safe and sensitive new detection methods. Gene Link’s primary focus is in the
development of facile non-radioactive detection methods. This product profile of Gene Link’s current gene detection product
line spotlights chemiluminescent detection based products as well as conventional radioactive based methods. The gene
detection systems can be divided into five broad groups based on detection methods:
- 35 -
Genemer™: Genemer™ is comprised of a primer pair for PCR amplification of the CGG triple repeat spanning region and
visualization of the product by gel electrophoresis and ethidium bromide staining. This product contains one tube containing
10 nmol of forward and reverse lyophilized primer. The quantity supplied is sufficient for 400 regular 50µl PCR reactions. The
10 nmol of primer when dissolved in 50µl water will give a solution of 200 µMolar i.e. 200 pmol/µl.
PCRProber™ Products: PCRProber™ Alkaline Phosphatase labeled probe is for amplification and non-radioactive detection
of Fragile X CGG trinucleotide repeat region amplified PCR product. The PCRProber™ kit comprises of a primer pair for PCR
amplification of the Fragile X CGG trinucleotide repeat region followed by gel blot and chemiluminescent detection using the
alkaline phosphatase oligonucleotide probe.
GeneProber™ Products: A specific gene fragment probe for Southern blot based hybridization of genomic DNA. The
GeneProber™ is available unlabeled for radioactive based methods and labeled with digoxigenin for chemiluminescent
detection. One tube is supplied containing 500 ng of lyophilized GeneProber™ probe. The probe spans the Eco RI and Eag I
sites of the FMR-1 gene. The quantity supplied is sufficient for at least 5 random prime labeling reactions using 100ng for
each reaction. Gene Link recommends using 25ng probe for each labeling reaction.
GScan™ Kits: GScan™ kits contain optimized PCR amplification reagents and a wide selection of fluorescent-labeled primers
for genotyping after PCR using fluorescent genetic analyzer instrument(s). Included in these kits are ready to run control
samples of various repeats of the triple repeat disorder kit. These control samples are for calibration with the molecular
weight markers for accurate size determination of the amplified fragments. It is strongly recommended that the genotyping
be followed up by using Southern blot detection methods when two alleles are not clearly discernable. Kit includes sufficient
reagents for 100 reactions.
Genemer™ Gscan™ Control DNA: PCR amplified HEX labeled fragment of the mutation region of a particular gene for use
with gene or mutation specific Genemer™. These control DNA’s are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA. One tube is supplied containing 25 µl of lyophilized DNA segment of the specified
CGG repeat fragment spanning the CGG repeat.
Genemer™ Control DNA: These are cloned fragment of the mutation region of a particular gene for use with gene or
mutation specific Genemer™ products. These control DNA’s are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA. One tube is supplied containing 500 ng of lyophilized DNA segment of the specified
CGG repeat fragment spanning the CGG repeat. The quantity supplied is sufficient for 1000 regular 50 µl PCR reactions.
Results and Interpretation
Fragile X PCRProber™ Results
Fragile X PCR blot.
Lane 1 pre-mutation female;
30/60 CGG repeats.
Non-radioactive detection,
~2 hr. exposure.
Fragile X GeneProber™ Results
Fragile X southern blot.
Lane 1 affected female.
Lanes 2, 3 & 5 are normal
males.
Lane 4 normal female.
Non-radioactive detection,
~2hr. exposure
- 36 -
Fragile X GScan™ Results
Fragile X PCR amplification of human genomic DNA using GLFX GScan 6-Fam labeled kit.
Fragile X Molecular Analysis Results Interpretation
Clinical Category
Risk mutation will
become full
mutation in next
generation
Number of CGG
repeats
Size of fragment
Fragment Size
Normal
(male/female)
Female
Carrier with
small
amplification
Female
carrier with
significant
amplification
Female
carrier with
Large
amplification
Carrier male
with premutation
(NTM)
Full Mutatiom
(Male/Female)
Carrier
with
Fragile X
Mosaicism
0%
moderate
significant
high
0%
moderate to high
can vary
from 0100%
6-45
46-69
70-86
87-200
40-200
>200
18-135
138-207
210-258
260-600
120-600
>600
221-338
341-410
413-461
464-803
323-803
>803
40-200/
>200
120-600/
>600
323-803/
>803
- 37 -
References:
1. Nelson, D.L. (1993) Growth Genetics and Hormone. 9:1-4.
2. Rousseau, F. et al. (1991) NEJM 325:1673-1681.
3. Verkerk, A. et al. (1991) Cell 65:905-914
4. Fu, Y.H et al. (1991) Cell 67:1047-1058.
5. Oberle, I. et al. (1991) Science 252:1097-1102.
6. Yu, S. et al. (1991) Science 252: 1179-1181.
7. Nelson, D.L. (1996) Growth Gen. and Hormone. 12:1-4.
8. Richards, R and Sutherland, G.R (1992) TIG 8: 249-255.
Ordering Information
Product
Size
Catalog No.
Price, $
Fragile X GScan™ TET Kit
1 Kit
40-2004-15TT
650.00
Fragile X GScan™ HEX Kit
1 Kit
40-2004-15HX
650.00
Fragile X GScan™ 6-FAM Kit
1 Kit
40-2004-15FM
650.00
Fragile X GScan™ Cy3 Kit
1 Kit
40-2004-15C3
650.00
Fragile X GScan™ Cy5 Kit
1 Kit
40-2004-15C5
650.00
500 ng
40-2004-40
350.00
110 µl
40-2004-41
400.00
Fragile X GeneProber™ GLFX1 Probe unlabeled
Fragile X CGG triple repeat spanning region unlabeled probe for radioactive
labeling and Southern blot detection. Suitable for random primer labeling.
Fragile X GeneProber™ GLFXDig1 Probe Digoxigenin labeled
Fragile X CGG triple repeat spanning region digoxigenin labeled probe for non-radioactive
Southern blot detection.
Fragile X PCRProber ™ AP labeled probe
Alkaline phosphatase labeled probe
Fragile X PCRProber ™ Kit for non-radioactive detection
Kit for performing non-radioactive PCR amplification based detection.(50 rxns)
12 µl
40-2004-31
400.00
5 blots
[50 rxns]
40-2004-32
650.00
Fragile X Genemer™ (spanning CGG triple repeat region)
10 nmols
40-2004-10
100.00
Kit for amplification and radioactive detection of Fragile X CGG triple repeat region amplified PCR
products using 35S or 32P.
1 Kit
40-2004-20
350.00
GLFX ~16 CGG repeat GScan Genemer™ Control DNA
25 µl
40-2004-01HX
175.00
GLFX ~29 CGG repeat GScan Genemer™ Control DNA
25 µl
40-2004-02HX
175.00
GLFX ~40 CGG repeat GScan Genemer™ Control DNA
25 µl
40-2004-03HX
175.00
GLFX Genemer™ Kit for Radioactive Detection
GLFX ~16 CGG repeat Genemer™ Control DNA
500 ng
40-2004-01
175.00
GLFX ~29 CGG repeat Genemer™ Control DNA
500 ng
40-2004-02
175.00
GLFX ~40 CGG repeat Genemer™ Control DNA
500 ng
40-2004-03
175.00
GLFX ~60 CGG repeat Genemer™ Control DNA
500 ng
40-2004-04
175.00
GLFX ~90 CGG repeat Genemer™ Control DNA
500 ng
40-2004-05
175.00
- 38 -
Huntington’s Disease
- 39 -
- 40 -
Huntington’s Disease
Background
Huntington disease (HD) is an autosomal dominant, progressive neurodegenerative disorder with a prevalence rate of about
5-10 affected persons per 100,000 in most western populations. The disorder presents with motor impairment, cognitive
deterioration, and psychiatric symptoms.
HD is caused by a CAG trinucleotide expansion within the first exon of the ITI5 gene on chromosome 4p16. The expanded
CAG repeats are translated into a polyglutamine tract in the Huntington protein, which is believed to cause a dominant gain of
function, leading to neuronal dysfunction and neurodegeneration.
The number of CAG repeats correlates inversely with the age of onset of symptoms. The American College of Medical
Genetics/American Society of Human Genetics/ Huntington Disease Genetics Testing Working Group divided
genotype/phenotype correlation in the following four categories for CAG repeat lengths:
•
•
•
•
Normal allele, ≤ 26 CAG repeats, generating a normal phenotype;
Intermediate allele, 27-35 CAG repeats, mutable normal allele generating a normal phenotype;
HD allele with reduced penetrance, 36-39 CAG repeats, generating a normal or HD phenotype;
HD allele, ≥ 40 CAG repeats, generating a HD phenotype.
The CAG trinucleotide expansion is unstable and can lengthen during transmission from parents to offspring. Thus, the sage
of onset can decrease from one generation to the next, a phenomenon known as anticipation. HD anticipation is more intense
in paternal transmission.
Molecular Analysis
The detection of expansion of a region of DNA sequence can be detected by PCR and Southern blotting procedures. These
methods can be used for all disorders involving increase in size of a region of DNA. DNA analysis for direct detection of CAG
expansion in Huntington Disease is based on enzymatic amplification of a fragment containing the CAG repeat sequence in
exon I of the HD gene. This test detects the CAG expansion by the size of the amplified product; an increase in size is
correlated with the corresponding number of CAG repeats and a calculated risk factor. Normal individuals have repeat
numbers of up to 30, while individuals with a high probability of developing HD carry more than 37 repeats. Individuals with
30-37 repeats have a high probability of passing on repeats in the pathological size range.
Polymerase Chain Reaction (PCR) based methods are fundamentally similar. The two primers are constructed such that they
span the region of the CAG trinucleotide repeat region. PCR is the most common method used to estimate the number of CAG
repeats. Since the CAG repeats in the HD gene are immediately 5’ of a CCG repeat which is also polymorphic in length, the
PCR product of this primer pair excludes the known adjacent polymorphic CCG repeat that can contribute to an inaccurate
determination of HD gene CAG repeat sizes in individuals who may have an HD gene CAG repeat allele close to the
normal/affected boundary.
Table 2 lists the size of PCR fragment in basepairs (bp) that can be expected when using the CAG primer mix F that has been
provided. The formula for determining PCR fragment size is 186 + 3n, where n= the number of CAG repeats.
Amplification of CCG and CAG + CCG Regions
Proximal to the 3’ end of the CAG trinucleotide repeat region is a second polymorphism that consists of a short sequence of 712 CCG trinucleotide repeats. As the presence of a second polymorphism would complicate the estimation of the CAG
expansion, primers that amplify the CAG trinucleotide repeat region have been carefully designed to exclude the CCG
trinucleotide repeat. However, when only a single allele is detected during amplification of the CAG repeat, inclusion of the
CCG polymorphism becomes useful. Detection of a single allele could result from one of the following situations A.) the
individual is homozygous for the CAG repeat; B.) a mutation in the region of primer binding precludes amplification of one
allele; C.) one allele contains a very large CAG expansion that is not amenable to PCR amplification.
Situations A and B can usually be resolved by amplification of the CAG + CCG region. Individuals that are homozygous for the
CAG repeat may not be homozygous for the CCG repeat, thus allowing for detection of the second allele. To verify whether
the individual is heterozygous for the CCG repeat, a primer mix for amplification of the CCG repeat region has been included
in the kit.
- 41 -
For situation B, the mutations that interfere with primer binding have been shown to occur primarily in the 3’ region of the
CAG repeat and affect the reverse CAG primer. The reverse primer used for amplification of the CAG + CCG region binds to
the DNA downstream from the mutable area and results in detection of the second allele.
In situation C both CAG and CAG + CCG amplification would detect only one allele. Detection of a second allele would be
possible by amplification of the CCG region, but only if the individual were heterozygous for the CCG polymorphism. In the
case of very large CAG expansions it is probably best to perform analysis by Southern blotting.
Table 1. HD CAG Fragment F Expected Length *
CAG(n)
Fragment Size (bp)
CAG(n)
Fragment Size (bp)
1
189
36
294
2
192
37
297
3
195
38
300
4
198
39
303
5
201
40
306
6
204
45
321
7
207
50
336
8
210
55
351
9
213
60
366
10
216
65
381
11
219
70
396
12
222
75
411
13
225
80
426
14
228
85
441
15
231
90
456
16
234
95
471
17
237
100
486
18
240
105
501
19
243
110
516
20
246
115
531
21
249
120
546
22
252
125
561
23
255
130
576
24
258
135
591
25
261
140
606
26
264
145
621
27
267
150
636
28
270
155
651
29
273
160
666
30
276
165
681
31
279
170
696
32
282
175
711
33
285
180
726
34
288
185
741
35
291
190
756
*Size of PCR fragment in basepairs (bp) that can be expected when using the CAG primer mix F that has been
provided. The formula for determining PCR fragment size is 186 + 3n, where n= the number of CAG repeats
- 42 -
*Table 2. HD CCG Fragment G Expected Length
(CCG)n
Fragment Size (bp)
7
163
8
166
9
169
10
172
11
175
12
178
*Above table lists the size of PCR fragment in base pairs (bp) that can be expected
when using the CCG repeat region primer mix G that has been provided. The formula
for determining PCR fragment size is 142 + 3n, where n= the number of CCG repeats.
Table 3..Trinucleotide Repeats in Human Genetic Disease
Repeata
Normal Lengthb
Fragile XA (FRAXA)
Fragile XE (FRAXE)
Fragile XF(FRAXF)
FRA16A
Jacobsen Syndrome (FRA11B)
Kennedy Syndrome (SMBA)
Myotonic Dstrophy (DM)
(CGG)n
(CCG)n
(CGG)n
(CCG)n
(CGC)n
(CAG)n
(CTG)n
6-52
4-39
7-40
16-49
11
14-32
5-37
Intermediate Length
(Premulation)a,b
59-230
? (31-61)
?
?
80
?
50-80
Huntington disease (HD)
Spinocerebellar ataxia 1 (SCA1)
Spinocerebellar ataxia 2 (SCA2)
Spinocerebellar ataxia 3 (SCA3)
/Machado Joseph disease (MJD)
Spinocerebellar ataxia 6 (SCA6)
Spinocerebellar ataxia 7 (SCA7)
Haw River syndrome (HRS; also
DRPLA)
Friedreich ataxia (FRDA)
(CAG)n
(CAG)n
(CAG)n
(CAG)n
10-34
6-39
14-31
13-44
36-39
None Reported
None Reported
None Reported
230-2,000
200-900
306-1,008
1,000-1,900
100-1,000
40-55
80-1,000; congenital,
2,000-3,000
40-121
40-81
34-59
60-84
(CAG)n
(CAG)n
(CAG)n
4-18
7-17
7-25
None Reported
28-35
?
21-28
38-130
49-75
(GAA)n
6-29
? (>34-40)
200-900
Disease
Full Disease Lengthb
Typically, repeats tracts contain sequence interruptions. See Pearson and Sinden (1998a) for a discussion of the sequence interruptions.
b No. of triplet repeats.
c A question mark (?) indicates potential mutagenic intermediate length, and an ellipsis (…) indicates none. Not all disease are associated with a
permutation length repeats tract or permutation disease condition.
a
- 43 -
*Table 4. HD CAG + CCG Fragment H Expected Length
(CCG)n
(CAG)n
7
8
9
10
11
12
Fragment Size (bp)
5
208
211
214
217
220
223
10
223
226
229
232
235
238
15
238
241
244
247
250
253
20
253
256
259
262
265
268
25
268
271
274
277
280
283
30
283
286
289
292
295
298
35
298
301
304
307
310
313
40
313
316
319
322
325
328
45
328
331
334
337
340
343
50
343
346
349
352
355
358
55
358
361
364
367
370
373
60
373
376
379
382
385
388
65
388
391
394
397
400
403
70
403
406
409
412
415
418
75
418
421
424
427
430
433
80
433
436
439
442
445
448
85
448
451
454
457
460
463
90
463
466
469
472
475
478
95
478
481
484
487
490
493
100
493
496
499
502
505
508
105
508
511
514
517
520
523
110
523
526
529
532
535
538
115
538
541
544
547
550
553
120
553
556
559
562
565
568
125
568
571
574
577
580
583
130
583
586
589
592
595
598
135
598
601
604
607
610
613
140
613
616
619
622
625
628
145
628
631
634
637
640
643
150
643
646
649
652
655
658
155
658
661
664
667
670
673
160
673
676
679
682
685
688
165
688
691
694
697
700
703
170
703
706
709
712
715
718
175
718
721
724
727
730
733
180
733
736
739
742
745
748
185
748
751
754
757
760
763
190
763
766
769
772
775
778
195
778
781
784
787
790
793
200
793
796
799
802
805
808
*Above table lists the size of PCR fragment in base pairs (bp) that can be expected
when using the primer mix H that amplifies the region that includes both the CAG and
CCG repeats. The formula for determining PCR fragment size is 172 + 3(CAG)n +
3(CCG)n, where n is the number of trinucleotide repeats.
Detection Methods
The choice of using methods for mutation analysis is restricted by our knowledge of the existence of a mutation and its type
and in parallel to the development of safe and sensitive new detection methods. Gene Link’s primary focus is in the
development of facile non-radioactive detection methods. This product profile of Gene Link’s current gene detection product
line spotlights chemiluminescent detection based products as well as conventional radioactive based methods. The gene
detection systems can be divided into five broad groups based on detection methods:
Genemer™: Genemer™ is comprised of a primer pair for PCR amplification of the CAG triple repeat spanning region and
visualization of the product by gel electrophoresis and ethidium bromide staining. This product contains one tube containing
10 nmol of forward and reverse lyophilized primer. The quantity supplied is sufficient for 400 regular 50µl PCR reactions. The
10 nmol of primer when dissolved in 50µl water will give a solution of 200 µMolar i.e. 200 pmol/µl.
- 44 -
Genemer™ Kit: The Genemer™ kit is a complete easy-to-use kit for reliable genotyping of a gene fragment. This line of
products is PCR based. The product includes a specific primer pair for gene or mutation specific amplification, optimized
buffers and dNTPs and in most cases, control DNA. Kit includes sufficient reagents for 100 detections.
PCRProber™ Products: PCRProber™ Alkaline Phosphatase labeled probe is for amplification and non-radioactive detection
of CAG trinucleotide repeat region amplified PCR product. The PCRProber™ kit comprises of a primer pair for PCR amplification
of the CAG trinucleotide repeat region followed by gel blot and chemiluminescent detection using the alkaline phosphatase
oligonucleotide probe.
GeneProber™: GeneProber™ is a specific gene fragment probe for Southern blot based hybridization of genomic DNA. The
GeneProber™ is available unlabeled for radioactive based methods and labeled with **digoxigenin for chemiluminescent
detection. One tube is supplied containing 500 ng of lyophilized GeneProber™ probe. The quantity supplied is sufficient for at
least 5 random prime labeling reactions using 100ng for each reaction. Gene Link recommends using 25ng probe for each
labeling reaction.
GScan™ Kits: GScan™ kits contain optimized PCR amplification reagents and a wide selection of fluorescent-labeled primers
for genotyping after PCR using fluorescent genetic analyzer instrument(s). Included in these kits are ready to run control
samples of various repeats of the triple repeat disorder kit. These control samples are for calibration with the molecular
weight markers for accurate size determination of the amplified fragments. It is strongly recommended that the genotyping
be followed up by using Southern blot detection methods when two alleles are not clearly discernable. Kit includes sufficient
reagents for 100 detections.
Genemer™ GScan™ Control DNA: PCR amplified HEX labeled fragment of the mutation region of a particular gene for use
with gene or mutation specific Genemer™. These control DNAs are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA. One tube is supplied containing 25 µl of lyophilized DNA segment of the specified CAG
repeat fragment spanning the CAG repeat.
Genemer™ Control DNA: These are cloned fragment of the mutation region of a particular gene for use with gene or
mutation specific Genemer™ products. These control DNAs are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA. One tube is supplied containing 500 ng of lyophilized DNA segment of the specified
CAG repeat fragment spanning the CAG repeat. The quantity supplied is sufficient for 1000 regular 50 µl PCR reactions.
Huntington’s Disease Control DNA with 134 CAG repeats
- 45 -
Results and Interpretation
The results obtained from the genetic analyzer will approximately show the fragment size amplified, based on these results an
interpretation can be made about the genotype of the sample. It is known that there is an overlap between the normal and
HD allele sizes. The repeat sizes obtained falling in the overlap region should be preferably repeated and possibly run with
more samples from other family members. Individuals with 36 repeats can be affected, and individuals with 36-39 repeats
can reach old age without developing HD. There is evidence that repeats in the 30-35 repeat range are prone to expansions
at meiosis, so it may be wise to suggest prenatal diagnosis, where appropriate, for individuals carrying such expansions.
References
1.
2.
Kremer, B et al. (1993) N. ENG. J. Med. 330: 1401-1406
The American College of Medical Genetica/American Society of Human Genetics Huntington Disease Genetic Testing
Working Group (1998) Am. J. Hum. Genet. 62: 000-000
3. Reiss O, Noerremoelle A, Soerensen SA, Epplen JT. Hum Mol Genet (1993) 2:
637-642.
4. Yu S, Fimmel A, Fung D, Trent RJ. Clin. Genet. (2000) 58: 469-472.
5. Williams LC, Hedge MR, Herrera G, Stapleton PM, Love DR. Mol. and Cell. Probes (1999) 13: 283-289.
- 46 -
Ordering Information
Product
Size
Catalog No.
Price, $
1 Kit
40-2025-15TT
650.00
Huntington Disease GScan™ HEX Kit
1 Kit
40-2025-15HX
650.00
Huntington Disease GScan™ 6-FAM Kit
1 Kit
40-2025-15FM
650.00
Huntington Disease GScan™ Cy3 Kit
1 Kit
40-2025-15C3
650.00
Huntington Disease GScan™ Cy5 Kit
1 Kit
40-2025-15C5
650.00
500 ng
40-2025-40
350.00
110 µl
40-2025-41
400.00
Huntington Disease GScan™ TET Kit
Huntington Disease GeneProber™ GLHD Probe unlabeled
Huntington Disease CAG triple repeat spanning region unlabeled probe for radioactive
labeling and Southern blot detection. Suitable for random primer labeling.
Huntington Disease GeneProber™ GLHD Probe Digoxigenin labeled
Huntington Disease CAG repeat spanning region digoxigenin labeled probe for nonradioactive detection Southern blot.
Huntington Disease PCRProber ™ AP labeled probe
Alkaline phosphatase labeled probe
Huntington Disease PCRProber ™ Kit for non-radioactive detection
Kit for performing non-radioactive PCR amplification based detection.(50 rxns)
12 µl
40-2025-31
400.00
5 blots
[50 rxns]
40-2025-32
650.00
Huntington Disease Genemer™ (spanning CAG triple repeat region)
10 nmols
40-2025-10
100.00
1 Kit
40-2025-11
250.00
Kit for amplification and radioactive detection of Huntington Disease CAG triple repeat region
amplified PCR products using 35S or 32P.
1 Kit
40-2025-20
350.00
GLHD 7 ~CAG repeat GScan Genemer™ Control DNA
25 µl
40-2025-05HX
175.00
GLHD 18 ~CAG repeat GScan Genemer™ Control DNA
25 µl
40-2025-01HX
175.00
GLHD 31 ~CAG repeat GScan Genemer™ Control DNA
25 µl
40-2025-07HX
175.00
GLHD 34 ~CAG repeat GScan Genemer™ Control DNA
25 µl
40-2025-02HX
175.00
GLHD 37 ~CAG repeat GScan Genemer™ Control DNA
25 µl
40-2025-08HX
175.00
GLHD 44 ~CAG repeat GScan Genemer™ Control DNA
25 µl
40-2025-03HX
175.00
GLHD 49 ~CAG repeat GScan Genemer™ Control DNA
25 µl
40-2025-09HX
175.00
GLHD 89 ~CAG repeat GScan Genemer™ Control DNA
25 µl
40-2025-04HX
175.00
GLHD 134 ~CAG repeat GScan Genemer™ Control DNA
25 µl
40-2025-61HX
175.00
GLHD 182 ~CAG repeat GScan Genemer™ Control DNA
25 µl
40-2025-62HX
175.00
GLHD 7 ~CAG repeat Genemer™ Control DNA
500 ng
40-2025-05
175.00
GLHD 18 ~CAG repeat Genemer™ Control DNA
500 ng
40-2025-01
175.00
GLHD 31 ~CAG repeat Genemer™ Control DNA
500 ng
40-2025-07
175.00
GLHD 34 ~CAG repeat Genemer™ Control DNA
500 ng
40-2025-02
175.00
GLHD 37 ~CAG repeat Genemer™ Control DNA
500 ng
40-2025-08
175.00
GLHD 44 ~CAG repeat Genemer™ Control DNA
500 ng
40-2025-03
175.00
GLHD 49 ~CAG repeat Genemer™ Control DNA
500 ng
40-2025-09
175.00
GLHD 89 ~CAG repeat Genemer™ Control DNA
500 ng
40-2025-04
175.00
GLHD 134 ~CAG repeat Genemer™ Control DNA
500 ng
40-2025-61
175.00
GLHD 182 ~CAG repeat Genemer™ Control DNA
500 ng
40-2025-62
175.00
Huntington Disease Genemer™ Kit (spanning CAG triple repeat region)
GLHD Genemer™ Kit for Radioactive Detection
- 47 -
- 48 -
Myotonic Dystrophy
- 49 -
- 50 -
Myotonic Dystrophy
Background
Myotonic dystrophy (Dystrophia Myotonica, DM) is the most common form of adult onset muscular dystrophy. It is an
autosomal dominant disorder with a prevalence of about 1 in 8000. The incidence varies from 1 in 475 in a region of Quebec
to about 1 in 25,000 in European populations and is extremely rare in African populations. Clinical expression is highly
variable and is related to age of onset. Onset of this disorder commonly occurs during young adulthood. However, it can occur
at any age and is extremely variable in degree of severity. Myotonic dystrophy affects skeletal muscle and smooth muscle, as
well as the eye, heart, endocrine system, and central nervous system. People with the mildest form of DM often go
undiagnosed and usually cataracts and minimal muscle involvement are the only visible sign of the condition. The classical
form of DM usually develops in early adult life and is characterized by progressive muscle stiffness and weakness.
Congenital DM (CDM) is the most severe form of the disease and is almost always inherited from affected mothers. It
presents in newborn babies who suffer from respiratory distress, hypotonia, motor and mental retardation and facial diplegia.
Diagnosis can be difficult if the family history is not known because muscle wasting may not be apparent and cataracts and
myotonia are absent. CDM patients who survive the neonatal period eventually learn to walk but 60-70% are mentally
retarded. By the age of 10 they develop myotonia and in adulthood they develop the additional complications associated with
adult onset disease.
Identification of the mutation in DM
The myotonic dystrophy gene locus and the underlying mutation were identified in 1992 (1-3). An expressed sequence called
cDNA25 was shown to detect a two-allele EcoRI polymorphism (8.6kb and 9.8kb) on Southern blots of normal individuals. It
also detects a larger variable fragment in DM patients, which can be up to 5kb longer than the larger, normal allele. When
this fragment is transmitted from an affected parent, it often increases in size, correlating well with the severity of the disease
in the affected child. The variable band can also show somatic heterogeneity in lymphocyte DNA that is seen as a diffuse
smear on a Southern blot. The EcoRI polymorphism is due to the insertion or deletion of consecutive Alu repeats 5 kb distal to
the unstable region – the 8.6kb allele contains two Alu repeats and the 9.8kb normal allele and the enlarged DM alleles are
associated with five Alu repeats. The discovery of unstable DNA at the DM locus provided an explanation for the phenomenon
of anticipation seen in DM. Sequence analysis of genomic clones spanning the expanded region revealed that the mutation
causing the instability is a trinucleotide repeat (CTG) which is highly polymorphic in the normal population and which
increases dramatically in length in DM patients.
Number of CTG repeats
Clinical Condition
Symptoms
5-27 repeats
Unaffected
Normal
50-100 repeats
Mild
cataracts, slight muscle problems later on in life
100-1000 repeats
Classical
myotonia, muscle wasting, premature balding,
gonadal atrophy, cardiac conduction defects
1000-4000 repeats
Congenital
hypotonia, mental retardation, facial diplegia
There are no definite repeat size boundaries for the three clinical groups and there are overlaps between the groups. A
trimodal distribution is observed in European populations, with (CTG)5 being the most frequently occurring allele, alleles of
11,12,13 and 14 make up the second mode and the final mode represents alleles of 19 and above.
Meiotic instability
The meiotic instability of the DM mutation has been shown to be dependent on the size of the parental repeat. For (CTG)n
repeats of <0.5kb a positive correlation between the size of the repeat and inter-generational enlargement was found equally
in male and female meioses but with CTG sequences of more than 0.5 kb observed that intergenerational variation was
greater through female meioses (4). The tendency for a repeat to undergo contraction was observed almost exclusively in
male meioses. It was found that the length of the CTG repeat expansion in DM patients was greater in DNA isolated from
muscle than in lymphocyte DNA (5). Rare cases have been reported where expansion of the CTG repeats is not seen in
individuals where the clinical symptoms are unequivocal and this may due to a deletion or point mutation as seen in some of
the other triplet repeat disorders such as fragile X syndrome.
The underlying mutations of DM are expansions of the CTG repeats located in the 3’ untranslated region (UTR) of the
myotonic dystrophy protein kinase (DMPK) gene on chromosome 19q. Severity of the disease is correlated with the length of
the repeat expansion. Normal individuals have from 5 to 30 repeat copies; mildly affected persons have at least 50 repeats,
while more severely affected patients have expansion of the repeat-containing segment up to several kilobase pairs.
Expansion is frequently observed in parent-to-child transmission, but extreme expansions are not transmitted through the
male line. This explains: 1.) the occurrence of the severe congenital form is almost exclusively in the offspring of affected
women; 2.) anticipation is commonly observed in affected families, that is, the disease demonstrates earlier onset and
greater severity in each successive generation. The overall risk of having a congenitally affected child for any carrier woman is
about 10%. If the woman has clinical signs of the condition, the risk of congenital myotonic dystrophy in offspring is 40% and
this rises to 50% in subsequent pregnancies if an affected child has previously has been born.
- 51 -
Genotyping
Molecular diagnosis of Myotonic Dystrophy involves a combination of direct PCR analysis and Southern blotting tests to
determine the CTG-repeat number within the DMPK gene. PCR can identify CTG expansions between 5-200 CTG repeats.
With larger expansions, Southern blot analysis of restriction fragments can be used for an accurate measure of the repeat
size. Genomic DNA is digested with Bam HI or Pst I. The DNA blot is then hybridized with either GLDM1 or GLDM2 CTG repeat
specific DNA probe.
Trinucleotide Repeats in Human Genetic Disease
Repeata
Normal Lengthb
Fragile XA (FRAXA)
Fragile XE (FRAXE)
Fragile XF(FRAXF)
FRA16A
Jacobsen Syndrome (FRA11B)
Kennedy Syndrome (SMBA)
Myotonic Dstrophy (DM)
(CGG)n
(CCG)n
(CGG)n
(CCG)n
(CGC)n
(CAG)n
(CTG)n
6-52
4-39
7-40
16-49
11
14-32
5-37
Intermediate Length
(Premulation)a,b
59-230
? (31-61)
?
?
80
?
50-80
Huntington disease (HD)
Spinocerebellar ataxia 1 (SCA1)
Spinocerebellar ataxia 2 (SCA2)
Spinocerebellar ataxia 3 (SCA3)
/Machado Joseph disease (MJD)
Spinocerebellar ataxia 6 (SCA6)
Spinocerebellar ataxia 7 (SCA7)
Haw River syndrome (HRS; also
DRPLA)
Friedreich ataxia (FRDA)
(CAG)n
(CAG)n
(CAG)n
(CAG)n
10-34
6-39
14-31
13-44
36-39
None Reported
None Reported
None Reported
230-2,000
200-900
306-1,008
1,000-1,900
100-1,000
40-55
80-1,000; congenital,
2,000-3,000
40-121
40-81
34-59
60-84
(CAG)n
(CAG)n
(CAG)n
4-18
7-17
7-25
None Reported
28-35
?
21-28
38-130
49-75
(CAA)n
6-29
? (>34-40)
200-900
Disease
Full Disease Lengthb
Typically, repeats tracts contain sequence interruptions. See Pearson and Sinden (1998a) for a discussion of the sequence interruptions.
b No. of triplet repeats.
c A question mark (?) indicates potential mutagenic intermediate length, and an ellipsis (…) indicates none. Not all disease are associated with a
permutation length repeats tract or permutation disease condition.
a
Molecular Analysis
The direct analysis of CTG repeats in the DMPK gene (chromosomal locus 19q13) is clinically available. An increased number
of CTG repeats is identified in essentially 100% of patients with DM. The number of CTG repeats ranges from 5 to 37 in
normal alleles. GTG repeat lengths in the range from about 38 to 49 are considered "premutations." Persons with CTG
expansions in the premutation range have not been reported as having developed symptoms, but their children are at risk of
inheriting a larger repeat size. Persons with CTG repeat length greater than 50 are frequently symptomatic.
Myotonic Dystrophy genotyping can be done by direct PCR amplification of the CTG trinucleotide repeats region or by
Southern analysis. In most cases both methods are used to complement the results. Congenital mutations usually cannot be
identified by PCR and southern analysis is the preferred method to distinguish full mutations.
The size of the CTG repeats can be determined by PCR analysis and sizing preferably on a sequencing gel. The PCR products
can be either labeled with 35S or 32P followed by autoradiography. Another attractive alternate is to run a cold PCR reaction
followed by blotting and hybridization with an alkaline phosphatase conjugated probe for non-radioactive detection
Southern blot analysis for Myotonic Dystrophy mutation detection involves the cleavage of DNA with either Bam HI or Pst
Ienzyme This method detects the size of CTG repeats region by hybridization of probe GLDM1 or GLDM2 to DNA that has been
digested with the appropriate restriction enzyme and blotted onto a membrane. The CTG repeat in the normal range yields a
~1377 bp with Bam HI and a ~1136 bp with Pst I digested DNA.
Detection Methods
The choice of using methods for mutation analysis is restricted by our knowledge of the existence of a mutation and its type
and in parallel to the development of safe and sensitive new detection methods. Gene Link’s primary focus is in the
development of facile non-radioactive detection methods. This product profile of Gene Link’s current gene detection product
line spotlights chemiluminescent detection based products as well as conventional radioactive based methods. The gene
detection systems can be divided into five broad groups based on detection methods:
- 52 -
Genemer™: Genemer™ is comprised of a primer pair for PCR amplification of the CTG triple repeat spanning region and
visualization of the product by gel electrophoresis and ethidium bromide staining. This product contains one tube containing
10 nmol of forward and reverse lyophilized primer. The quantity supplied is sufficient for 400 regular 50µl PCR reactions. The
10 nmol of primer when dissolved in 50µl water will give a solution of 200 µMolar i.e. 200 pmol/µl.
Genemer™ Kit: The Genemer™ kit is a complete easy-to-use kit for reliable genotyping of a gene fragment. This line of
products is PCR based. The product includes a specific primer pair for gene or mutation specific amplification, optimized
buffers and dNTPs and in most cases, control DNA. Kit includes sufficient reagents for 100 detections.
PCRProber™ Products: PCRProber™ Alkaline Phosphatase labeled probe is for amplification and non-radioactive detection
of CTG trinucleotide repeat region amplified PCR product. The PCRProber™ kit comprises of a primer pair for PCR amplification
of the CTG trinucleotide repeat region followed by gel blot and chemiluminescent detection using the alkaline phosphatase
oligonucleotide probe.
GeneProber™: GeneProber™ is a specific gene fragment probe for Southern blot based hybridization of genomic DNA. The
GeneProber™ is available unlabeled for radioactive based methods and labeled with **digoxigenin for chemiluminescent
detection. One tube is supplied containing 500 ng of lyophilized GeneProber™ probe. The quantity supplied is sufficient for at
least 5 random prime labeling reactions using 100ng for each reaction. Gene Link recommends using 25ng probe for each
labeling reaction.
GScan™ Kits: GScan™ kits contain optimized PCR amplification reagents and a wide selection of fluorescent-labeled primers
for genotyping after PCR using fluorescent genetic analyzer instrument(s). Included in these kits are ready to run control
samples of various repeats of the triple repeat disorder kit. These control samples are for calibration with the molecular
weight markers for accurate size determination of the amplified fragments. It is strongly recommended that the genotyping
be followed up by using Southern blot detection methods when two alleles are not clearly discernable. Kit includes sufficient
reagents for 100 detections.
Genemer™ Gscan Control DNA: PCR amplified HEX labeled fragment of the mutation region of a particular gene for use
with gene or mutation specific Genemer™. These control DNAs are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA. One tube is supplied containing 25 µl of lyophilized DNA segment of the specified CTG
repeat fragment spanning the CTG repeat.
Genemer™ Control DNA: These are cloned fragment of the mutation region of a particular gene for use with gene or
mutation specific Genemer™ products. These control DNAs are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA. One tube is supplied containing 500 ng of lyophilized DNA segment of the specified
CTG repeat fragment spanning the CTG repeat. The quantity supplied is sufficient for 1000 regular 50 µl PCR reactions.
Myotonic Dystrophy Genemer Control DNA containg 129 CTG repeats
- 53 -
Results and Interpretation
- Amplified fragment of ~144bp contains 12 CTG repeats. Run control samples to compare results.
DM PCRProber™ Results
References:
1.
2.
3.
4.
5.
6.
7.
Fu YH, Pizzuti A, Fenwick RG Jr, King J, Rajnarayan S, Dunne PW, Dubel J, Nasser
GA, Ashizawa T, de Jong P, et al. (1992) An unstable triplet repeat in a gene related
to myotonic muscular dystrophy. Science 255: 1256-1258.
Aslanidis et al. (1992) Cloning of the essential myotonic dystrophy region and
mapping of the putative defect. Nature 355: 548-551.
Brook et al. (1992) Molecular basis of myotonic dystrophy: expansion of a
trinucleotide (CTG) repeat at the 3-prime end of a transcript encoding a protein
kinase family member. Cell 68: 799-808.
Lavedan et al. (1993) Myotonic dystrophy: size- and sex-dependent dynamics of
CTG meiotic instability, and somatic mosaicism. Am. J. Hum. Genet. 52: 875-883.
Anvret et al. ((1993) Larger expansions of the CTG repeat in muscle compared to
lymphocytes from patients with myotonic dystrophy. Human Molecular Genetics
2:1397-1400.
Mathieu J, Allard P, Potvin L, Prevost C, Begin P (1999) A 10-year study of mortality
in a cohort of patients with myotonic dystrophy. Neurology 52:1658-62
Redman JB, Fenwick RG Jr, Fu YH, Pizzuti A, Caskey CT (1993) Relationship
between parental trinucleotide GCT repeat length and severity of myotonic dystrophy
in offspring. JAMA 269:1960-5
- 54 -
Ordering Information
Size
Catalog No.
Price, $
Myotonic Dystrophy GScan™ TET Kit
Product
1 Kit
40-2026-15TT
650.00
Myotonic Dystrophy GScan™ HEX Kit
1 Kit
40-2026-15HX
650.00
Myotonic Dystrophy GScan™ 6-FAM Kit
1 Kit
40-2026-15FM
650.00
Myotonic Dystrophy GScan™ Cy3 Kit
1 Kit
40-2026-15C3
650.00
Myotonic Dystrophy GScan™ Cy5 Kit
1 Kit
40-2026-15C5
650.00
500 ng
40-2026-40
350.00
110 µl
40-2026-41
400.00
Myotonic Dystrophy GeneProber™ GLDM1 Probe unlabeled
Myotonic Dystrophy CTG triple repeat spanning region unlabeled probe for radioactive
labeling and Southern blot detection. Suitable for random primer labeling.
Myotonic Dystrophy GeneProber™ GLDMDig2 Probe Digoxigenin labeled.
Myotonic dystrophy CTG triple repeat spanning region digoxigenin labeled probe for
Southern blot non-radioactive detection of Pst I digested DNA.
Myotonic Dystrophy PCRProber ™ AP labeled probe
Alkaline phosphatase labeled probe
Myotonic Dystrophy PCRProber ™ Kit for non-radioactive detection
Kit for performing non-radioactive PCR amplification based detection.(50 rxns)
12 µl
40-2026-31
400.00
5 blots
[50 rxns]
40-2026-32
650.00
Myotonic Dystrophy Genemer™ (spanning CTG triple repeat region)
10 nmols
40-2026-10
100.00
1 Kit
40-2026-11
250.00
1 Kit
40-2026-20
350.00
GLDM 12 ~CTG repeat GScan Genemer™ Control DNA
25 µl
40-2026-01HX
175.00
GLDM 45 ~CTG repeat GScan Genemer™ Control DNA
25 µl
40-2026-02HX
175.00
GLDM 93 ~CTG repeat GScan Genemer™ Control DNA
25 µl
40-2026-03HX
175.00
GLDM 129 ~CTG repeat GScan Genemer™ Control DNA
25 µl
40-2026-04HX
175.00
Myotonic Dystrophy Genemer™ Kit (spanning CTG triple repeat region)
GLDM Genemer™ Kit for Radioactive Detection
Kit for amplification and radioactive detection of Myotonic Dystrophy CTG triple repeat region
amplified PCR products using 35S or 32P.
GLDM 182 ~CTG repeat GScan Genemer™ Control DNA
25 µl
40-2026-05HX
175.00
GLDM ~12 CTG repeat Genemer™ Control DNA
500 ng
40-2026-01
175.00
GLDM ~45 CTG repeat Genemer™ Control DNA
500 ng
40-2026-02
175.00
GLDM ~93 CTG repeat Genemer™ Control DNA
500 ng
40-2026-03
175.00
GLDM ~129 CTG repeat Genemer™ Control DNA
500 ng
40-2026-04
175.00
GLDM ~182 CTG repeat Genemer™ Control DNA
500 ng
40-2026-05
175.00
- 55 -
- 56 -
Friedreich’s Ataxia
- 57 -
- 58 -
Friedreich’s Ataxia
Background
Friedreich’s ataxia (FRDA [MIM 229300], NM_181425) is an autosomal recessive neurodegenerative disorder characterized by
a progressive loss of voluntary muscle coordination (ataxia). The disorder affects upper and lower limbs, and the head and
neck. FRDA is characterized clinically by progressive gait and limb ataxia; signs of upper motoneuron dysfunction including
dysarthria, areflexia, and loss of the senses of position and vibration; cardiomyopathy; diabetes mellitus; and secondary
skeletal abnormalities. Most patients develop hypertrophic cardiomyopathy and skeletal abnormalities, and some become
diabetic (1,2). These symptoms progress with age, such that most patients become wheelchair-bound by their late twenties
and die by their mid-thirties most commonly of congestive heart failure. Some of the other symptoms include muscle
weakness, loss of pressure and position sense in the arms and legs, speech problem and heart disease. Unlike some
neurological diseases, FRDA does not affect mental capacity. See recent reviews (3,4).
Although rare, FRDA is the most prevalent inherited ataxia, affecting about 1-2 in every 50,000 individuals. It is usually
diagnosed in childhood between the ages of 5 and 15. The majority (~98%) of patients with FRDA are homozygous for a GAA
repeat expansion within the first intron of frataxin gene. The remaining patients are compound heterozygotes for the GAA
expansion and for point mutations within the X25 gene. In normal alleles, the repeat varies in size between 7 and 30 units,
whereas in mutated alleles the repeat length ranges from 100 to more than 1000 units. Generally, the milder forms or late
onset of the disease are associated with shorter expansions.
FRDA is caused by degeneration of nerve tissue in the spinal cord and of nerves that extend to peripheral areas such as the
arms and legs. The disorder is associated with an unstable expansion of GAA repeats in the first intron of the FRDA gene,
called X25, on chromosome 9q13. The encoded protein, frataxin, is located in mitochondria and reduced in FRDA patients. It
is suggested that FRDA is the result of mitochondrial iron overload leading to excess production of free radicals, which results
in cellular damage and death.
The majority (>95%) of patients with FRDA are homozygous for large expansions of a GAA triplet repeat sequence (66 1800
triplets) located within the first intron of the gene X25, which encodes the protein frataxin (2). The expansion causes a severe
reduction in the levels of frataxin, a 210 amino acid protein that is targeted to mitochondrial matrix and that appears to play a
crucial role in iron homeostasis. The severity of the disease is directly correlated with the length of the expansion. A very small
minority of patients are compound heterozygotes for the GAA expansion and for point mutations within the X25 gene.
Chamberlain and coworkers have recently summarized all point mutations described to date (5).
Frataxin RNA levels were severely reduced lymphoblast cell lines of patients with FRDA who were homozygous for the GAA
expansion. Several groups have demonstrated that the GAA-repeat expansion interferes with transcription. It has been show
by various groups that the GAA Triplet Repeat Expansion acts as an Impediment to Transcription (3).
Genetically, FRDA belongs to a class of neurodegenerative disorders in which the underlying gene, FRDA, carries an unstable
trinucleotide-repeat sequence. At least eight other members of this class have been identified, including HD and many types of
spinocerebellar ataxia. However, key genetic features separate FA from the other trinucleotide-repeat disorders. First, the
sequence of the trinucleotide repeat in the FRDA1 gene is GAA (2), whereas a CAG repeat occurs in the other trinucleotideassociated ataxias, and other repeats (CTG or CGG) are seen in other trinucleotide diseases. Second, the GAA repeat of FRDA
is located in the first intron and is therefore noncoding, whereas the CAG repeat in HD and the spinocerebellar ataxias always
occurs within an exon and encodes glutamine.
The third difference is that FRDA is inherited in a recessive manner, and multiple lines of evidence suggest that loss of function
leads to the disease. In contrast, in the other trinucleotide-repeat disorders, whether the repeat occurs in an expressed DNA
sequence or in a 3' untranslated sequence, the mutation is inherited in a dominant manner, and it is a gain of function of the
affected protein or RNA that perturbs cell physiology.
The severity of the disease correlates with decreased FRDA expression and with the length of the hyperexpansive repeat.
Normally, this gene, which encodes the protein frataxin, contains <39 GAA repeats, but in patients with FRDA, this locus
contains 66 1,700 repeat units. This hyperexpansion results in marked decreases in frataxin mRNA levels, thought to result
from the formation of an unusual non-β DNA structure inhibiting transcription (3). More than 95% of patients with FRDA are
homozygous for the GAA hyperexpansion, although the alleles are polymorphic in the number of GAA repeats. Studies have
shown a correlation between the length of the GAA expansion on the smaller allele and severity of disease (1). An inverse
correlation between GAA expansion size and frataxin protein levels has been observed in lymphoblast cell lines from patients
with FRDA (3). Together, these findings suggest that lack of frataxin protein in critical tissues leads to FRDA. The remaining
5% of patients with FRDA are compound heterozygotes for the GAA expansion on one allele and carry point mutations within
FRDA1 on the other allele.
The most common disease-causing point mutation in frataxin is I154F (numbering based on the initiator methionine of the
predicted open reading frame [ORF]), prevalent in some southern Italian families. Those individuals carrying this missense
mutation on one allele, together with the hyperexpansion on the other allele, are indistinguishable in disease severity when
compared with homozygous relatives who carry the GAA triplet expansion on both alleles (4). Another missense mutation in
frataxin, G130V, compounded with a hyperexpansive allele, is associated with a milder and more slowly progressive disease
course (3).
- 59 -
Trinucleotide Repeats in Human Genetic Disease
Repeata
Normal Lengthb
Fragile XA (FRAXA)
Fragile XE (FRAXE)
Fragile XF(FRAXF)
FRA16A
Jacobsen Syndrome (FRA11B)
Kennedy Syndrome (SMBA)
Myotonic Dstrophy (DM)
(CGG)n
(CCG)n
(CGG)n
(CCG)n
(CGC)n
(CAG)n
(CTG)n
6-52
4-39
7-40
16-49
11
14-32
5-37
Intermediate Length
(Premulation)a,b
59-230
? (31-61)
?
?
80
?
50-80
Huntington disease (HD)
Spinocerebellar ataxia 1 (SCA1)
Spinocerebellar ataxia 2 (SCA2)
Spinocerebellar ataxia 3 (SCA3)
/Machado Joseph disease (MJD)
Spinocerebellar ataxia 6 (SCA6)
Spinocerebellar ataxia 7 (SCA7)
Haw River syndrome (HRS; also
DRPLA)
Friedreich ataxia (FRDA)
(CAG)n
(CAG)n
(CAG)n
(CAG)n
10-34
6-39
14-31
13-44
36-39
None Reported
None Reported
None Reported
230-2,000
200-900
306-1,008
1,000-1,900
100-1,000
40-55
80-1,000; congenital,
2,000-3,000
40-121
40-81
34-59
60-84
(CAG)n
(CAG)n
(CAG)n
4-18
7-17
7-25
None Reported
28-35
?
21-28
38-130
49-75
(GAA)n
6-29
? (>34-40)
200-900
Disease
Full Disease Lengthb
Typically, repeats tracts contain sequence interruptions. See Pearson and Sinden (1998a) for a discussion of the sequence interruptions.
No. of triplet repeats.
c A question mark (?) indicates potential mutagenic intermediate length, and an ellipsis (…) indicates none. Not all disease are associated with a
permutation length repeats tract or permutation disease condition.a
b
Meiotic instability and Somatic Variation in GAA Expansion Length
The GAA expansion shows intergenerational variation in length, with evidence for changes in the prezygotic and postzygotic
stages. Studies have shown that the expanded alleles seen in patients arose from a small pool of uninterrupted "large normal"
alleles referred to as "premutations." Interruptions within the pure GAA triplet repeats impeded these large normal alleles from
expanding into disease-causing alleles. De Michele et al. (10) have noted that premutation alleles can undergo large
expansions in a single generation. Expanded GAA repeats can expand or contract when transmitted through the female
germline. In contrast, contractions are favored in male transmission. This is attributed to postzygotic mechanisms, because
shorter expansions are seen in sperm DNA when compared with lymphocyte DNA (prezygotic mechanism). However, evidence
for postzygotic variation in repeat number has also been suggested, because the degree of repeat contraction in the sperm is
greater than that actually seen in intergenerational transmission and because the overall length of expanded alleles is shorter
in homozygous versus heterozygous carriers. The formation of unexpected parallel duplex has been shown in GAA and TTC
trinucleotide repeats of Friedreich's ataxia (11). This presumably interferes with normal transcription activity.
Genotyping
Molecular diagnosis of Friedreich’s Ataxia is available. It involves a combination of direct PCR analysis and Southern blotting
tests to determine the GAA-repeat number within the FRDA gene. PCR can identify GAA expansions between 5-200 GAA
repeats. With larger expansions, Southern blot analysis of restriction fragments can be used for an accurate measure of the
repeat size. Genomic DNA is double digested with Pst I and Bgl II. The DNA blot is then hybridized with FRDA-GL3 DNA probe.
Molecular Analysis
The direct analysis of GAA repeats in the FRDA gene (chromosomal locus 9q13) is clinically available. An increased number of
GAA repeats is identified in essentially 100% of patients with FRDA. The number of GAA repeats ranges from 5 to <30 in
normal alleles. GAA repeat lengths in the range from about >30-49 are considered "premutations." Persons with GAA
expansions in the premutation range have not been reported as having developed severe symptoms, but their children are at
risk of inheriting a larger repeat size. Persons with GAA repeat length greater than 50 are frequently symptomatic.
Friedreich’s Ataxia genotyping can be done by direct PCR amplification of the GAA trinucleotide repeats region or by Southern
analysis. In most cases both methods are used to complement the results. Full mutations usually cannot be identified by PCR
and southern analysis is the preferred method to distinguish full mutations.
- 60 -
The size of the GAA repeats can be determined by PCR analysis and sizing preferably on a sequencing gel. The PCR products
can be either labeled with 35S or 32P followed by autoradiography. Another attractive alternate is to run a cold PCR reaction
followed by blotting and hybridization with an alkaline phosphatase conjugated probe for non-radioactive detection
Southern blot analysis for Friedreich’s Ataxia mutation detection involves the cleavage of genomic DNA with Pst I and Bgl II
enzyme This method detects the size of GAA repeats region by hybridization of probe FRDA-GL3 to DNA that has been
digested with the appropriate restriction enzyme and blotted onto a membrane. The GAA repeat in the normal range yields a
~1084 bp.
Detection Methods
The choice of using methods for mutation analysis is restricted by our knowledge of the existence of a mutation and its type
and in parallel to the development of safe and sensitive new detection methods. Gene Link’s primary focus is in the
development of facile non-radioactive detection methods. This product profile of Gene Link’s current gene detection product
line spotlights chemiluminescent detection based products as well as conventional radioactive based methods. The gene
detection systems can be divided into five broad groups based on detection methods:
Genemer™: Genemer™ is comprised of a primer pair for PCR amplification of the CAA triple repeat spanning region and
visualization of the product by gel electrophoresis and ethidium bromide staining. This product contains one tube containing
10 nmol of forward and reverse lyophilized primer. The quantity supplied is sufficient for 400 regular 50µl PCR reactions. The
10 nmol of primer when dissolved in 50µl water will give a solution of 200 µMolar i.e. 200 pmol/µl.
Genemer™ Kit: The Genemer™ kit is a complete easy-to-use kit for reliable genotyping of a gene fragment. This line of
products is PCR based. The product includes a specific primer pair for gene or mutation specific amplification, optimized
buffers and dNTPs and in most cases, control DNA. Kit includes sufficient reagents for 100 detections.
PCRProber™ Products: PCRProber™ Alkaline Phosphatase labeled probe is for amplification and non-radioactive detection
of CAA trinucleotide repeat region amplified PCR product. The PCRProber™ kit comprises of a primer pair for PCR amplification
of the CAA trinucleotide repeat region followed by gel blot and chemiluminescent detection using the alkaline phosphatase
oligonucleotide probe.
GeneProber™: GeneProber™ is a specific gene fragment probe for Southern blot based hybridization of genomic DNA. The
GeneProber™ is available unlabeled for radioactive based methods and labeled with **digoxigenin for chemiluminescent
detection. One tube is supplied containing 500 ng of lyophilized GeneProber™ probe. The quantity supplied is sufficient for at
least 5 random prime labeling reactions using 100ng for each reaction. Gene Link recommends using 25ng probe for each
labeling reaction.
GScan™ Kits: GScan™ kits contain optimized PCR amplification reagents and a wide selection of fluorescent-labeled primers
for genotyping after PCR using fluorescent genetic analyzer instrument(s). Included in these kits are ready to run control
samples of various repeats of the triple repeat disorder kit. These control samples are for calibration with the molecular
weight markers for accurate size determination of the amplified fragments. It is strongly recommended that the genotyping
be followed up by using Southern blot detection methods when two alleles are not clearly discernable. Kit includes sufficient
reagents for 100 detections.
Genemer™ Gscan Control DNA: PCR amplified HEX labeled fragment of the mutation region of a particular gene for use
with gene or mutation specific Genemer™. These control DNAs are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA. One tube is supplied containing 25 µl of lyophilized DNA segment of the specified CAA
repeat fragment spanning the CAA repeat.
Genemer™ Control DNA: These are cloned fragment of the mutation region of a particular gene for use with gene or
mutation specific Genemer™ products. These control DNAs are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA. One tube is supplied containing 500 ng of lyophilized DNA segment of the specified
CAA repeat fragment spanning the CAA repeat. The quantity supplied is sufficient for 1000 regular 50 µl PCR reactions.
Results and Interpretation
- Amplified fragment of ~150bp contains 10 GAA repeats.
- Run control samples to compare results.
Number of GAA repeats
Clinical Condition
Symptoms
5-30 repeats
Unaffected
Normal
?34-40 repeats
Mild
Premutation
200-900 repeats
Severe
Full mutation
- 61 -
References
1. Durr, A, Cossee M, Agid Y, Campuzano V, Mignard C, Penet C, Mandel JL, et al (1996) Clinical and genetic
abnormalities in patients with Friedreich's ataxia. New Engl J Med 335:1169 1175.
2. Campuzano V, Montermini L, Moltó MD, Pianese L, Cossée M, Cavalcanti F, Monros E, et al (1996) Friedreich's ataxia:
autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 271:1423 1427.
3. Patel PI and Grazia Isaya G (2001) Friedreich Ataxia: From GAA Triplet Repeat Expansion to Frataxin Deficiency. Am. J.
Hum. Genet., 69:15-24.
4. Simon A. B. Knight, S.A.B; Kim, R;Pain,D and Dancis,A. (1999) The Yeast Connection to Friedreich Ataxia. Am. J. Hum.
Genet., 64:365-371.
5. Monrόs, E. et al. (1997) Am. J. Hum. Genet. 61: 101-110.
6. Castro, M. et al. (2000) Hum. Genet. 106: 86-92.
7. Pook MA, Al Mahdawi SA, Thomas NH, Appleton R, Norman A, Mountford R, Chamberlain S (2000) Identification of
three novel frameshift mutations in patients with Friedreich's ataxia. J Med Genet 37:E38.
8. Bradley JL, Blake JC, Chamberlain S, Thomas PK, Cooper JM, Schapira AH (2000) Clinical, biochemical and molecular
genetic correlations in Friedreich's ataxia. Hum Mol Genet 9:275 282.
9. Ohshima K, Montermini L, Wells RD, Pandolfo M (1998) Inhibitory effects of expanded GAA.TTC triplet repeats from
intron I of the Friedreich ataxia gene on transcription and replication in vivo. J Biol Chem 273:14588 14595
10. Bidichandani SI, Ashizawa T, Patel PI (1998) The GAA triplet-repeat expansion in Friedreich ataxia interferes with
transcription and may be associated with an unusual DNA structure. Am J Hum Genet 62:111 121.
11. Bradley JL, Blake JC, Chamberlain S, Thomas PK, Cooper JM, Schapira AH (2000) Clinical, biochemical and molecular
genetic correlations in Friedreich's ataxia. Hum Mol Genet 9:275 282.
12. De Michele G, Cavalcanti F, Criscuolo C, Pianese L, Monticelli A, Filla A, Cocozza S (1998) Parental gender, age at
birth and expansion length influence GAA repeat intergenerational instability in the X25 gene: pedigree studies and
analysis of sperm from patients with Friedreich's ataxia. Hum Mol Genet 7:1901 1906
13. LeProust EM, Pearso CE, Sinden RR, Gao X (2000) Unexpected formation of parallel duplex in GAA and TTC
trinucleotide repeats of Friedreich's ataxia. J Mol Biol 302:1063 1080.
- 62 -
Ordering Information
Product
Size
Catalog No.
Price, $
1 Kit
40-2027-15TT
650.00
Friedreich’s Ataxia GScan™ HEX Kit
1 Kit
40-2027-15HX
650.00
Friedreich’s Ataxia GScan™ 6-FAM Kit
1 Kit
40-2027-15FM
650.00
Friedreich’s Ataxia GScan™ Cy3 Kit
1 Kit
40-2027-15C3
650.00
Friedreich’s Ataxia GScan™ Cy5 Kit
1 Kit
40-2027-15C5
650.00
500 ng
40-2027-40
350.00
110 µl
40-2027-41
400.00
Friedreich’s Ataxia GScan™ TET Kit
Friedreich’s Ataxia GeneProber™ FRDA-GL3 Probe unlabeled
Friedreich’s Ataxia GAA triple repeat spanning region unlabeled probe for radioactive
labeling and Southern blot detection. Suitable for random primer labeling.
Friedreich Ataxia GeneProber™ FRDA-GL3 Probe Digoxigenin labeled
Friedreich Ataxia GAA triple repeat spanning region digoxigenin labeled probe for Southern
blot non-radioactive detection.
Friedreich’s Ataxia PCRProber ™ AP labeled probe
Alkaline phosphatase labeled probe
Friedreich’s Ataxia PCRProber ™ Kit for non-radioactive detection
Kit for performing non-radioactive PCR amplification based detection.(50 rxns)
12 µl
40-2027-31
400.00
5 blots
[50 rxns]
40-2027-32
650.00
Friedreich’s Ataxia Genemer™ (spanning GAA triple repeat region)
10 nmols
40-2027-10
100.00
1 Kit
40-2027-11
250.00
Kit for amplification and radioactive detection of Friedreich’s Ataxia GAA triple repeat region
amplified PCR products using 35S or 32P.
1 Kit
40-2027-20
350.00
GLFRDA ~64 GAA repeat GScan™ Genemer Control DNA
25 µl
40-2027-01HX
175.00
GLFRDA ~102 GAA repeat GScan™ Genemer Control DNA
25 µl
40-2027-02HX
175.00
GLFRDA ~110 GAA repeat GScan™ Genemer Control DNA
25 µl
40-2027-03HX
175.00
GLFRDA ~125 GAA repeat GScan™ Genemer Control DNA
25 µl
40-2027-04HX
175.00
GLFRDA ~9 GAA repeat Gscan™ Genemer Control DNA
25 µl
40-2027-05HX
175.00
GLFRDA ~64 GAA repeat Genemer™ Control DNA
500 ng
40-2027-01
175.00
GLFRDA ~102 GAA repeat Genemer™ Control DNA
500 ng
40-2027-02
175.00
GLFRDA ~110 GAA repeat Genemer™ Control DNA
500 ng
40-2027-03
175.00
GLFRDA ~125 GAA repeat Genemer™ Control DNA
500 ng
40-2027-04
175.00
GLFRDA ~9 GAA repeat Genemer™ Control DNA
500 ng
40-2027-05
175.00
Friedreich’s Ataxia Genemer™ Kit (spanning GAA triple repeat region)
FRDA Genemer™ Kit for Radioactive Detection
- 63 -
- 64 -
Spinal and Bulbar Muscular Atrophy (SBMA)/Kennedy’s Disease
- 65 -
- 66 -
Spinal and Bulbar Muscular Atrophy (SBMA)/Kennedy’s Disease
Background
Spinal and bulbar muscular atrophy (SBMA), also called Kennedy’s disease, is an X-linked form of motor neuron disease that
affects male only. The disease is adult onset and characterized by progressive muscle wasting, loss of motor neurons in the
spinal cord and brainstem, and partial androgen insensitivity.
SBMA is caused by a dynamic mutation in the first exon of the androgen receptor gene, involving a CAG trinucleotide repeat.
The CAG repeat encodes a run of glutamine residues near the amino terminus of the protein, which is involved in the
modulation of transcription activation. While the expansion mutation causes some loss of transcriptional activity by the
androgen receptor, the predominant effect of expansion is probably a toxic gain of function, similar to the mechanism of other
polyglutamine expansion diseases.
The trinucleotide repeat of exon 1 is polymorphic in the normal population, with the size varying between 11 and 33. In
patients with SBMA, the expanded repeat ranges in number from 38 to 72. As with the other repeat expansion diseases, the
longer the repeat the earlier the onset of the disease.
SBMA occurs in less than 1/50,000 live male births and appears to be much more common in the Japanese population than in
any other ethnic group due to a founder effect.
Table 3..Trinucleotide Repeats in Human Genetic Disease
Repeata
Normal Lengthb
Fragile XA (FRAXA)
Fragile XE (FRAXE)
Fragile XF(FRAXF)
FRA16A
Jacobsen Syndrome (FRA11B)
Kennedy Syndrome (SMBA)
Myotonic Dstrophy (DM)
(CGG)n
(CCG)n
(CGG)n
(CCG)n
(CGC)n
(CAG)n
(CTG)n
6-52
4-39
7-40
16-49
11
14-32
5-37
Intermediate Length
(Premulation)a,b
59-230
? (31-61)
?
?
80
?
50-80
Huntington disease (HD)
Spinocerebellar ataxia 1 (SCA1)
Spinocerebellar ataxia 2 (SCA2)
Spinocerebellar ataxia 3 (SCA3)
/Machado Joseph disease (MJD)
Spinocerebellar ataxia 6 (SCA6)
Spinocerebellar ataxia 7 (SCA7)
Haw River syndrome (HRS; also
DRPLA)
Friedreich ataxia (FRDA)
(CAG)n
(CAG)n
(CAG)n
(CAG)n
10-34
6-39
14-31
13-44
36-39
None Reported
None Reported
None Reported
230-2,000
200-900
306-1,008
1,000-1,900
100-1,000
40-55
80-1,000; congenital,
2,000-3,000
40-121
40-81
34-59
60-84
(CAG)n
(CAG)n
(CAG)n
4-18
7-17
7-25
None Reported
28-35
?
21-28
38-130
49-75
(GAA)n
6-29
? (>34-40)
200-900
Disease
Full Disease Lengthb
Typically, repeats tracts contain sequence interruptions. See Pearson and Sinden (1998a) for a discussion of the sequence interruptions.
b No. of triplet repeats.
c A question mark (?) indicates potential mutagenic intermediate length, and an ellipsis (…) indicates none. Not all disease are associated with a
permutation length repeats tract or permutation disease condition.
a
Genotyping
SBMA can be rapidly diagnosed by performing PCR amplification of the CAG repeat region of the androgen gene, followed by
agarose gel electrophoresis of the amplified fragments to determine their size. The SBMA Genemer flanks the CAG repeat and
generates a PCR product of 222 + 3n bp (n = number of CAG repeats). This test can be used to identify affected individuals,
heterozygote females and pre-symptomatic males.
- 67 -
Detection Methods
The choice of using methods for mutation analysis is restricted by our knowledge of the existence of a mutation and its type
and in parallel to the development of safe and sensitive new detection methods. Gene Link’s primary focus is in the
development of facile non-radioactive detection methods. This product profile of Gene Link’s current gene detection product
line spotlights chemiluminescent detection based products as well as conventional radioactive based methods. The gene
detection systems can be divided into five broad groups based on detection methods:
Genemer™: Genemer™ is comprised of a primer pair for PCR amplification of the amplification of the fragment of interest
and visualization of the product by gel electrophoresis and ethidium bromide staining. This product contains one tube
containing 10 nmol of forward and reverse lyophilized primer. The quantity supplied is sufficient for 400 regular 50µl PCR
reactions. The 10 nmol of primer when dissolved in 50µl water will give a solution of 200 µMolar i.e. 200 pmol/µl.
Genemer™ Kit: The Genemer™ kit is a complete easy-to-use kit for reliable genotyping of a gene fragment. This line of
products is PCR based. The product includes a specific primer pair for gene or mutation specific amplification, optimized
buffers and dNTPs and in most cases, control DNA. Kit includes sufficient reagents for 100 detections.
PCRProber™ Products: PCRProber™ Alkaline Phosphatase labeled probe is for amplification and non-radioactive detection
of CAG trinucleotide repeat region amplified PCR product. The PCRProber™ kit comprises of a primer pair for PCR amplification
of the CAG trinucleotide repeat region followed by gel blot and chemiluminescent detection using the alkaline phosphatase
oligonucleotide probe.
GeneProber™: GeneProber™ is a specific gene fragment probe for Southern blot based hybridization of genomic DNA. The
GeneProber™ is available unlabeled for radioactive based methods and labeled with **digoxigenin for chemiluminescent
detection. One tube is supplied containing 500 ng of lyophilized GeneProber™ probe. The quantity supplied is sufficient for at
least 5 random prime labeling reactions using 100ng for each reaction. Gene Link recommends using 25ng probe for each
labeling reaction.
GScan™ Kits: GScan™ kits contain optimized PCR amplification reagents and a wide selection of fluorescent-labeled primers
for genotyping after PCR using fluorescent genetic analyzer instrument(s). Included in these kits are ready to run control
samples of various repeats of the triple repeat disorder kit. These control samples are for calibration with the molecular
weight markers for accurate size determination of the amplified fragments. It is strongly recommended that the genotyping
be followed up by using Southern blot detection methods when two alleles are not clearly discernable. Kit includes sufficient
reagents for 100 detections.
Genemer™ Gscan Control DNA: PCR amplified HEX labeled fragment of the mutation region of a particular gene for use
with gene or mutation specific Genemer™. These control DNAs are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA. One tube is supplied containing 25 µl of lyophilized DNA segment of the specified CAG
repeat fragment spanning the CAG repeat.
Genemer™ Control DNA: These are cloned fragment of the mutation region of a particular gene for use with gene or
mutation specific Genemer™ products. These control DNAs are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA. One tube is supplied containing 500 ng of lyophilized DNA segment of the specified
CAG repeat fragment spanning the CAG repeat. The quantity supplied is sufficient for 1000 regular 50 µl PCR reactions.
Results and Interpretation
Normal individuals have ≤33 CAG repeats. For an individual with 20 CAG repeats, a 288-bp PCR product would be expected
from the PCR reaction. A general formula, 222 + 3n bp, can be applied to calculate other sizes of CAG repeats, where n
represents the number of CAG repeats.
References:
1. H MacLean, et al. (1996) Journal of the Neurological Sciences 135: 149-157.
2. S Igarashi, et al. (1992) Neurology 42(12): 2300-2302.
- 68 -
Ordering Information
Product
Size
Catalog No.
Price, $
500 ng
40-2032-40
350.00
Kennedy Disease (SBMA) GeneProber™ GLSBMA Probe unlabeled
Kennedy Disease CAG triple repeat spanning region unlabeled probe for radioactive labeling and
Southern blot detection. Suitable for random primer labeling.
Kennedy Disease (SBMA) GeneProber™ GLSBMA Probe Digoxigenin labeled
Kennedy Disease CAG triple repeat spanning region digoxigenin labeled probe for non-radioactive
Southern blot detection.
CAG repeat PCRProber ™ Kit (spanning triple repeat region)
Kit for performing PCR amplification and chemiluminescent based detection for all CAG triple repeat
disorders.
CAG repeat PCRProber ™ AP labeled probe
Alkaline phosphatase labeled probe
110 µl
40-2032-41
400.00
5 blots
[50 rxns]
40-20XX-32
650.00
12 µl
40-20XX-31
400.00
Kennedy Disease Genemer™
10 nmols
40-2032-10
100.00
1 Kit
40-2032-11
250.00
Kit for amplification and radioactive detection of Kennedy Disease CAG triple repeat region amplified PCR
products using 35S or 32P.
1 Kit
40-2032-20
350.00
Kennedy Disease GScan™ TET Kit
1 Kit
40-2032-15TT
650.00
Kennedy Disease GScan™ HEX Kit
1 Kit
40-2032-15HX
650.00
Kennedy Disease GScan™ 6-FAM Kit
1 Kit
40-2032-15FM
650.00
Kennedy Disease GScan™ Cy3 Kit
1 Kit
40-2032-15C3
650.00
Kennedy Disease GScan™ Cy5 Kit
1 Kit
40-2032-15C5
650.00
Kennedy (SBMA) ~23 CAG repeat GScan Genemer™ Control DNA
25 µl
40-2032-01HX
175.00
Kennedy Disease (SBMA) ~23 CAG repeat Genemer™ Control DNA
500 ng
40-2032-01
175.00
Kennedy Disease (SBMA) Genemer™ Kit
Kennedy Disease Genemer™ Kit for Radioactive Detection
- 69 -
- 70 -
Rh
(RhD gene exon 10 specific)
(Rh Ee and Cc exon 7 specific)
- 71 -
- 72 -
Rh
(RhD gene exon 10 specific)
(Rh Ee and Cc exon 7 specific)
Background
The Rh based blood grouping is termed positive or negative based on the presence or absence of the D antigen. Rh
alloimmunization in Rh negative pregnant women is of concern because of the potential for the fetus to develop hemolytic
disease of newborns and autoimmune diseases. Anemia leading to hydrops, perinatal death, or both occurs in 25% of fetuses
sensitized to the RhD antigen in the absence of optimal management. In utero diagnosis and treatment considerably improves
the condition with survival rates greater than 75% in severely affected fetuses. However these invasive therapies may be
unnecessary in some cases if the fetal Rh status were known prenatally.
A method of determining fetal Rh status early in pregnancy is now possible by DNA analysis of amniotic cells (1). The Rh
blood group locus consists of two related structural genes, D and CcEe. These highly homologous genes which share greater
than 96% identity in their coding region have been cloned and the molecular basis of the Rh blood group established (2). The
RhD-positive and RhD-negative polymorphism is associated with the presence or the absence of the D gene (there is no ‘d’
gene). The C/c and E/e antigens are encoded by a unique gene. The E/e associated nucleotide polymorphism results in one
amino acid substitution at position 226 (proline to alanine), whereas the C/c antigenic polymorphism consists of six nucleotide
substitutions leading to four amino acid changes at position 16 (Cys16Trp), 60 (Ile60Leu), 68 (Ser68Asn) and 103
(Ser103Pro).
DNA analysis for Rh genotype loci specifically amplifies DNA fragments for the RhD and RhCcEe gene. Due to the high
sensitivity and specificity of the test at the DNA level, occasionally the results may not match those obtained by serologic
methods. The test will type individuals as RhD positive who are Du low grade status serologically (3-5), this is due to the
absence of the gene product at the protein level due to partial deletions. The total error rate should be less than 1%.
Detection Methods
The choice of using methods for mutation analysis is restricted by our knowledge of the existence of a mutation and its type
and in parallel to the development of safe and sensitive new detection methods. Gene Link’s primary focus is in the
development of facile non-radioactive detection methods. This product profile of Gene Link’s current gene detection product
line spotlights chemiluminescent detection based products as well as conventional radioactive based methods. The gene
detection systems can be divided into five broad groups based on detection methods:
Genemer™: Genemer™ is comprised of a primer pair for PCR amplification of amplification of the fragment of interest and
visualization of the product by gel electrophoresis and ethidium bromide staining. This product contains one tube containing
10 nmol of forward and reverse lyophilized primer. The quantity supplied is sufficient for 400 regular 50 µl PCR reactions. The
10 nmol of primer when dissolved in 50µl water will give a solution of 200 µMolar i.e. 200 pmol/µl.
Genemer™ Kit: The Genemer™ kit is a complete easy-to-use kit for reliable genotyping of a gene fragment. This line of
products is PCR based. The product includes a specific primer pair for gene or mutation specific amplification, optimized
buffers and dNTPs and in most cases, control DNA. Kit includes sufficient reagents for 100 detections.
- 73 -
Genemer™ Control DNA: These are cloned fragment of the mutation region of a particular gene for use with gene or
mutation specific Genemer™ products. These control DNAs are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA. One tube is supplied containing 500 ng of lyophilized DNA segment of the specified
region. The quantity supplied is sufficient for 1000 regular 50 µl PCR reactions.
Results and Interpretation
The primers A1/A2 will give 136 bp PCR product specific for Rh EeCc gene exon 7 (Catalog No. 40-2003-10) and primers
A3/A4 will give 186 bp PCR product specific for Rh D gene exon 10 (Catalog No. 40-2002-10). Can perform multiplex of A1/A2
& A3/A4 as shown in the figure below.
Figure. Lane 1 is molecular weight markers. Lanes 2-4 represent PCR
from a Rh D positive DNA and Lanes 5-7 represent PCR from Rh D
negative DNA. Lanes 2, 5 & 7 PCR product of A1/A2 amplification,
lanes 3 from A3/A4 amplification. Lane 4 is a multiplex of A1/A2 and
A3/A4 amplification. RhD negative- 136 bp only;
RhD positive- both 136 & 186 bp.
References
1. Bennet, P.R., et al. (1993) Prenatal determination of fetal RhD type by DNA amplification. NEJM 329:607-610.
2. Mouro, I., et al. (1993) Molecular genetic basis of the human Rhesus blood group system. Nature Genetics 5:62-65.
3. Simsek, S., Bleeker, P.M., Borne, A.. E.G. (1994) Prenatal determination of fetal RhD type. NEJM 330:795.
4. Bennet, P., Warwick,R. and Carton, J-P. (1994) Prenatal determination of fetal RhD type. NEJM 330:795-796.
5. Westhoff,C.M. and Wylie, D.E. (1994) Identification of a new RhD-specific mRNA from K562 cells. Blood 84:3098-3100 by
the polymerase chain reaction. Hum. Genet. 82:271-274
*The polymerase chain reaction (PCR) process is covered by patents owned by Hoffmann-La Roche. A license to perform is automatically granted by the use of authorized reagents.
Ordering Information
Product
Size
Catalog No.
Price, $
RhD (RhD gene exon 10 specific) Genemer™
10 nmol
40-2002-10
100.00
Rh EeCc (Rh Ee and Cc exon 7 specific) Genemer™
10 nmol
40-2003-10
100.00
RhD (RhD gene exon 10 specific) Genemer™ Kit
1 Kit
40-2002-11
200.00
Rh EeCc (Rh Ee and Cc exon 7 specific) Genemer™ Kit
1 Kit
40-2003-11
200.00
RhD (RhD gene exon 10 specific) Genemer™ Control DNA
500 ng
40-2002-01
115.00
Rh EeCc (Rh Ee and Cc exon 7 specific) Genemer™ Control DNA
500 ng
40-2003-01
115.00
- 74 -
SRY, X and Y
- 75 -
- 76 -
SRY, X and Y
Background
The human sex determining region on the Y chromosome has been identified and the gene has been termed as SRY.
Mutations in the SRY gene have been found in XY females. Sex reversal in XY females results from the failure of the testis
determination or differentiation pathways. Some XY females with gonadal dysgenesis have lost the SRY gene from the Y
chromosome by terminal exchange between the sex chromosome or by other deletions or mutations affecting activity (1,2).
DNA analysis for a specific region of SRY together with alphoid repeat regions of the X and Y chromosome is used for accurate
sex determination (in the absence of mutations involving SRY), and in the characterization of X-linked genetic diseases, Y
chromosome anomalies such as XY females with gonadal dysgenesis, and for XO/XY mosaicism in patients with Turner
syndrome. The DNA test involves the amplification of specific regions of X, Y and SRY. The presence of amplified product
directly indicates the presence of the cognate DNA fragments on the chromosome. Normal XX females will amplify only X
chromosome specific fragment showing double intensity as compared with amplification from normal XY male. SRY and Y
fragments will only be amplified from individuals with a Y chromosome.
Normal PCR amplified fragment size
SRY
422 bp
X chromosome
130 bp
Y chromosome
170 bp
Detection Methods
The choice of using methods for mutation analysis is restricted by our knowledge of the existence of a mutation and its type
and in parallel to the development of safe and sensitive new detection methods. Gene Link’s primary focus is in the
development of facile non-radioactive detection methods. This product profile of Gene Link’s current gene detection product
line spotlights chemiluminescent detection based products as well as conventional radioactive based methods. The gene
detection systems can be divided into five broad groups based on detection methods:
Genemer™: Genemer™ is comprised of a primer pair for PCR amplification of amplification of the fragment of interest and
visualization of the product by gel electrophoresis and ethidium bromide staining. This product contains one tube containing
10 nmol of forward and reverse lyophilized primer. The quantity supplied is sufficient for 400 regular 50 µl PCR reactions. The
10 nmol of primer when dissolved in 50µl water will give a solution of 200 µMolar i.e. 200 pmol/µl.
Genemer™ Kit: The Genemer™ kit is a complete easy-to-use kit for reliable genotyping of a gene fragment. This line of
products is PCR based. The product includes a specific primer pair for gene or mutation specific amplification, optimized
buffers and dNTPs and in most cases, control DNA. Kit includes sufficient reagents for 100 detections.
Genemer™ Control DNA: These are cloned fragment of the mutation region of a particular gene for use with gene or
mutation specific Genemer™ products. These control DNAs are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA. One tube is supplied containing 500 ng of lyophilized DNA segment of the specified
region. The quantity supplied is sufficient for 1000 regular 50 µl PCR reactions.
Results and Interpretation
-Normal female DNA should only amplify X specific fragment.
-Normal male DNA should amplify all fragments (SRY, X & Y)
Figure 1. SRY, X and Y PCR amplification gel profile. Lane 1,
molecular weight marker. Lanes 2-4 male DNA, lanes 5-7
female DNA. Lanes 2 and 5 SRY amplification, lanes 3 and 6 X
amplification, lanes 4 and 7 Y amplification. Note the absence
of amplification of SRY and Y from female DNA (lanes 5 & 7).
- 77 -
Figure 2. A screen shot from a Cepheid Real Time PCR using SRY and X Genemer™ specific TaqMan and Molecular Beacons
References
1.
1.
2.
Berta et al. (1990) Genetic evidence equating SRY and the testis-determining factor. Nature 348:448-451.
Jager et al. (1990) A human XY female with frame shift mutation in the candidate testis-determining gene SRY.
Nature 348:452-453.
Witt, M. & Erickson, R.P. (1989) A rapid method for detection of Y-chromosome DNA from dried blood specimens by
the polymerase chain reaction. Hum. Genet. 82:271-274.
- 78 -
Ordering Information
Product
Size
Catalog No.
Price, $
10 nmols
40-2020-10
100.00
X alphoid repeat Genemer™
10 nmols
40-2021-10
100.00
Y alphoid repeat Genemer™
10 nmols
40-2022-10
100.00
SRY (sex determining region on Y) Genemer™ Kit
1 Kit
40-2020-11
200.00
X alphoid repeat Genemer™ Kit
1 Kit
40-2021-11
200.00
SRY (sex determining region on Y) Genemer™
Y alphoid repeat Genemer™ Kit
1 Kit
40-2022-11
200.00
SRY (sex determining region on Y) Genemer™ Control DNA
500 ng
40-2020-01
115.00
X alphoid repeat Genemer™ Control DNA
500 ng
40-2021-01
115.00
Y alphoid repeat Genemer™ Control DNA
500 ng
40-2022-01
115.00
- 79 -
- 80 -
Sickle Cell
- 81 -
- 82 -
Sickle Cell
Background
Sickle cell anemia is an autosomal recessive disease. The hemoglobin beta, delta and gamma chain genes are on Chromosome 11 and
the alpha chains are coded on Chromosome 16. The beta variants such as Hb S, Hb C, and Hb D all occur from mutations on
Chromosome 11. The cause of the disorder sickle cell anemia is due to a single base change of A to T in the β globin chain
resulting in the substitution of amino acid glutamine to valine at the sixth position. The resulting mutant globin chain is
termed as the Hb S. Hemoglobin S is freely soluble when fully oxygenated, under conditions of low oxygen tension the red
cells become grossly abnormal assuming a sickle shape leading to aggregation and hemolysis. Homozygous Hb S is a serious
hemoglobinopathy found almost exclusively in the Black population. About 8% of American Blacks are carriers and about
0.2% are affected. Heterozygotes (sickle cell trait) are clinically normal, although their red cells will sickle when subjected to very low oxygen pressure
in vitro.
Hemoglobin C (Hb C) is due to a single base change of G to A leading to a substitution of lysine for glutamic acid in the sixth
position of the β globin chain. Hb C occurs in higher frequency in individuals with heritage from Western Africa, Italy, Greece,
Turkey, and the Middle East. There is shortened red cell survival in Hb C homozygotes and sickling complications in compound
heterozygotes for Hb S and Hb C.
Molecular Analysis
DNA analysis for the sickle cell mutation is done by specific amplification of the DNA region spanning the mutation using polymerase chain reaction followed
by enzymatic cleavage of the amplified product. Sickle cell mutation abolishes a restriction endonuclease site (Dde I). Electrophoretic resolution of the
fragment pattern reveals the presence or absence of the mutation. Clear genotyping of normal, carrier and homozygous DNA is achieved.
Sequence Information
Hb-A: …TCCTGAGGAG…
Hb-S: …TCCTGTGGAG…
Hb-C: …TCCTAAGGAG…
Detection Methods
The choice of using methods for mutation analysis is restricted by our knowledge of the existence of a mutation and its type
and in parallel to the development of safe and sensitive new detection methods. Gene Link’s primary focus is in the
development of facile non-radioactive detection methods. This product profile of Gene Link’s current gene detection product
line spotlights chemiluminescent detection based products as well as conventional radioactive based methods. The gene
detection systems can be divided into five broad groups based on detection methods:
Genemer™: Genemer™ is comprised of a primer pair for PCR amplification of amplification of the fragment of interest and
visualization of the product by gel electrophoresis and ethidium bromide staining. This product contains one tube containing
10 nmol of forward and reverse lyophilized primer. The quantity supplied is sufficient for 400 regular 50 µl PCR reactions. The
10 nmol of primer when dissolved in 50µl water will give a solution of 200 µMolar i.e. 200 pmol/µl.
Genemer™ Kit: The Genemer™ kit is a complete easy-to-use kit for reliable genotyping of a gene fragment. This line of
products is PCR based. The product includes a specific primer pair for gene or mutation specific amplification, optimized
buffers and dNTPs and in most cases, control DNA. Kit includes sufficient reagents for 100 detections.
Genemer™ Control DNA: These are cloned fragment of the mutation region of a particular gene for use with gene or
mutation specific Genemer™ products. These control DNAs are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA. One tube is supplied containing 500 ng of lyophilized DNA segment of the specified
region. The quantity supplied is sufficient for 1000 regular 50 µl PCR reactions.
- 83 -
Results And Interpretation
Mutation abolishes restriction site.
PCR Product Fragment Size 233 bp
Fragment Sizes After Dde I Digestion
A/A
A/S
S/S
178+55 bp
233+178+55 bp
233 bp
Figure 1. Typical Sickle cell genotype analysis of PCR product
digested with Dde I. Lane 1 is molecular weight markers. Lane 2
is undigested PCR product. Lanes 3, 4 and 6 is DNA with A/S
geneotype. Lane 5 is A/A genotype DNA and Lane 7 represents
DNA with S/S genotype.
References:
Saiki et al. (1985) Science 230:1350-1354
Wu et al. (1989) PNAS 86:2757-2760
Conner et al. (1983) PNAS 80:278-282
1.
2.
3.
Ordering Information
Product
Size
Catalog No.
Price, $
10 nmols
40-2001-10
100.00
1 Kit
40-2001-11
200.00
Sickle Cell HbA Genemer™ Control DNA
500 ng
40-2001-01
115.00
Sickle Cell HbS Genemer™ Control DNA
500 ng
40-2001-02
115.00
Sickle Cell HbC Genemer™ Control DNA
500 ng
40-2001-03
115.00
Sickle Cell Genemer™
Sickle Cell Genemer™ Kit
- 84 -
Cystic Fibrosis
- 85 -
- 86 -
Cystic Fibrosis
Background
Cystic Fibrosis (CF) is the most common recessive disorder affecting Caucasians of European descent with a carrier frequency
of 1 in 25. The frequency of these mutations is given below in the table on the left. Mutations at the CF locus occur in other
racial and ethnic groups as shown below in the table on the right. Genemers™ are available for the five most frequent
mutation listed.
CF Mutation Frequency
Caucasian Non-Jewish
Ashkenazic Jews
Mutation
Frequency
Mutation
Frequency
75.8%
W1282X
60%
∆F508
G551D
3.2%
23%
∆F508
G542X
2.7%
G542X
4%
R553X
1.4%
N1303K
4%
N1303K
1.4%
3849+10kb
4%
C→T
Total
~84%
Total
~95%
Frequency of CF Carriers
Caucasian Americans of European
1 in 25
descent
Ashkenazic Jews
1 in 29
Hispanic Americans
1 in 45
African Americans
1 in 60
Asian Americans
1 in 150
Detection Methods
The choice of using methods for mutation analysis is restricted by our knowledge of the existence of a mutation and its type
and in parallel to the development of safe and sensitive new detection methods. Gene Link’s primary focus is in the
development of facile non-radioactive detection methods. This product profile of Gene Link’s current gene detection product
line spotlights chemiluminescent detection based products as well as conventional radioactive based methods. The gene
detection systems can be divided into five broad groups based on detection methods:
Genemer™: Genemer™ is comprised of a primer pair for PCR amplification of amplification of the fragment of interest and
visualization of the product by gel electrophoresis and ethidium bromide staining. This product contains one tube containing
10 nmol of forward and reverse lyophilized primer. The quantity supplied is sufficient for 400 regular 50 µl PCR reactions. The
10 nmol of primer when dissolved in 50µl water will give a solution of 200 µMolar i.e. 200 pmol/µl.
Genemer™ Kit: The Genemer™ kit is a complete easy-to-use kit for reliable genotyping of a gene fragment. This line of
products is PCR based. The product includes a specific primer pair for gene or mutation specific amplification, optimized
buffers and dNTPs and in most cases, control DNA. Kit includes sufficient reagents for 100 detections.
Genemer™ Control DNA: These are cloned fragment of the mutation region of a particular gene for use with gene or
mutation specific Genemer™ products. These control DNAs are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA. One tube is supplied containing 500 ng of lyophilized DNA segment of the specified
region. The quantity supplied is sufficient for 1000 regular 50 µl PCR reactions.
Results and Interpretation
Mutation
PCR Product
∆F508
G542X
G551D/R553X
N1303K
114
114
60
Bst I
Hind II
Bst NI
N/N
fragment size(s),
bp
79/79
90+24
59+55
40+20
W1282X
473
Mnl I
178+172+123
CT3849
437
Hph I
349+88
Restriction
Enzyme
N/M
fragment size(s),
bp
79/76
114+90+24
114+59+55
60+40+20
301+178+172+12
3
349+222+127+88
M/M
fragment size(s),
bp
76/76
114
114
114
301+ 172
222+127+88
References:
Kerem et al. (1990) PNAS. 87: 8447-8451 (caucasian mutation primers)
Abeliovich et. al (1992) AJHG 51:951-956 (CT+10kb mutation)
Shoshani et al. (1992) AJHG 50: 222-228 (W1282X,mutation)
Ng et al. (1991) Hum. Genet. 87:613-617
- 87 -
Ordering Information
Product
Size
Catalog No.
Price, $
Cystic Fibrosis ∆F508 Genemer™
10 nmols
40-2029-11
100.00
Cystic Fibrosis G542X Genemer™
10 nmols
40-2010-12
100.00
Cystic Fibrosis W1282X Genemer™
10 nmols
40-2011-13
100.00
Cystic Fibrosis R553X Genemer™
10 nmols
40-2012-14
100.00
Cystic Fibrosis G551D Genemer™
10 nmols
40-2013-15
100.00
Cystic Fibrosis CT3849+10kb Genemer™
10 nmols
40-2014-16
100.00
Cystic Fibrosis N1303K Genemer™
10 nmols
40-2015-17
100.00
*The polymerase chain reaction (PCR) process is covered by patents owned by Hoffmann-La Roche. A license to perform is automatically granted by the use of authorized reagents.
- 88 -
Tay Sachs Disease
- 89 -
- 90 -
Tay Sachs Disease
Background
Tay-Sachs disease (TSD) is an autosomal recessive disorder caused by several different mutations in the HEX A gene,
encoding the α-subunit of β-hexosaminidase A (Hex A). Tay-Sachs disease, which is prevalent in the Ashkenazi Jewish
population with a 3% carrier frequency, is the prototype of human GM2-gangliosidosis due to the defective α-subunit.
Diminished catabolism of GM2 ganglioside leads to the lysosomal accumulation of the undegraded glycolipid in neurons.
Clinically the disease presents in varying degrees of severity as related to the extent of hexosaminidase A deficiency.
Tay-Sachs disease is classified as classic infantile or adult-onset. This classification relates to the type of mutation present.
Two mutations found in the infantile TSD are the Exon 11 and the Intron 12 mutations, together representing 91% of TSD
mutations. Both of these mutations lead to deficiency of α-chain mRNA. The mutation in exon 11 4 bp insertion, leads to a
nonsense mutation 9 bases from the insertion site. This would theoretically lead to a truncated protein product, and in fact no
mRNA is detected. In the intron 12 mutation, the splice junction site is altered causing aberrant splicing and leading to the
deficiency of α-chain mRNA. Both these mutations result in little or no protein product and the clinical phenotype of classic
infantile Tay-Sachs disease.
The adult-onset TSD mutation in Exon 7 is a point mutation (G to A), leading to the substitution of amino acid glycine at
position 269 for serine (G269S). This mutation affects the active site causing a drastic reduction in catalytic activity which
results in the delayed appearance of TSD. The exon 7 mutation is relatively rare and accounts for about 3% of TSD patients.
Pseudodeficiency is used to denote healthy individuals who have deficient Hex A enzymatic activity when synthetic substrates
are used, but normal activity with the natural substrate. The mutation responsible for pseudodeficiency has been identified.
Compound heterozygotes with a pseudodeficiency allele and other mutant alleles have been identified.
Mutations 1-3 in the table constitute approximately 62% of the non-Jewish mutations; 4-5 constitute approximately 38% of
the non-Jewish mutations.
DNA analysis for the following four mutations are performed for the Ashkenazic Jewish population. The frequency of these
mutations is given below.
TAY-SACHS MUTATION ANALYSIS
Mutation
1277insTATC (Exon 11, 4 bp insertion; infantile
classic)
1421+1G→C (Intron12, splice junction;
infantile)
Frequency (Jewish)
73%
18%
G269S (Exon 7, G→A; adult onset)
3%
R247W (739C→T, pseudodeficiency allele)
2%
R249W (745C→T, pseudodeficiency allele)
3% (non-Jewish)
Detection Methods
The choice of using methods for mutation analysis is restricted by our knowledge of the existence of a mutation and its type
and in parallel to the development of safe and sensitive new detection methods. Gene Link’s primary focus is in the
development of facile non-radioactive detection methods. This product profile of Gene Link’s current gene detection product
line spotlights chemiluminescent detection based products as well as conventional radioactive based methods. The gene
detection systems can be divided into five broad groups based on detection methods:
Genemer™: Genemer™ is comprised of a primer pair for PCR amplification of amplification of the fragment of interest and
visualization of the product by gel electrophoresis and ethidium bromide staining. This product contains one tube containing
10 nmol of forward and reverse lyophilized primer. The quantity supplied is sufficient for 400 regular 50 µl PCR reactions. The
10 nmol of primer when dissolved in 50µl water will give a solution of 200 µMolar i.e. 200 pmol/µl.
Genemer™ Kit: The Genemer™ kit is a complete easy-to-use kit for reliable genotyping of a gene fragment. This line of
products is PCR based. The product includes a specific primer pair for gene or mutation specific amplification, optimized
buffers and dNTPs and in most cases, control DNA. Kit includes sufficient reagents for 100 detections.
Genemer™ Control DNA: These are cloned fragment of the mutation region of a particular gene for use with gene or
mutation specific Genemer™ products. These control DNAs are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA. One tube is supplied containing 500 ng of lyophilized DNA segment of the specified
region. The quantity supplied is sufficient for 1000 regular 50 µl PCR reactions.
- 91 -
Interpretation of Results
Mutation
1277insTATC (Exon 11, 4 bp insertion;
infantile classic)
1421+1G→C (Intron12, splice junction;
infantile)
G269S (Exon 7, G→A; adult onset)
R247W (739C→T, pseudodeficiency
allele)
PCR Product
Normal
Homo
159
159
163
135
120+15
85+35+15
190
75+59+34+16+8
73+67+34+16
161
146+15
105+41+15
Carrier
159+163
120+85+35+15
All (Carrier & homo)
146+105+41+15
*The polymerase chain reaction (PCR) process is covered by patents owned by Hoffmann-La Roche. A license to perform is automatically granted by the use of authorized reagents.
References:
1. Myerowitz,R. (1988) PNAS 85:3955-3959. ( Intron 12, splice junction)
2. Myerowitz, R. & Costigan, F.C. (1988) JBC 263:18587-18589. (Exon 11, 4 bp insertion)
3. Ruth, N. & Proia, R. (1989) Science 243:1471-1475. (Exon 7, G269S)
4. Paw,B., Kaback,M. & Neufeld, E. (1989) PNAS 86:2413-2417. (Exon 7, G269S)
5. Grebner, E. & Tomczak, J. (1991) AJHG 48:604-607. (Distribution of mutations)
6.Triggs-Raine et al. (1992) AJHG 51:793-801. (Pseudodeficiency allele)
Ordering Information
Size
Catalog No.
Price, $
Tay-Sachs disease 1277insTATC Genemer™
Product
10 nmols
40-2028-11
100.00
Tay-Sachs disease 1421+1G→C Genemer™
10 nmols
40-2028-12
100.00
Tay-Sachs disease G269S Genemer™
10 nmols
40-2028-13
100.00
Tay-Sachs disease R247W Genemer™
10 nmols
40-2028-14
100.00
- 92 -
Gaucher’s Disease
- 93 -
- 94 -
Gaucher’s Disease
Introduction
Gaucher disease (GD) is caused by deficient activity of the lysosomal enzyme glucosylceramidase and the resultant
accumulation of its undegraded substrate, glucosylceramide (GL1) and other glycolipids.. Gaucher disease is suspected in
individuals with characteristic bone involvement, visceral and hematologic changes, or CNS involvement. Mutation analysis of
the GBA gene (chromosomal locus 1q21) is available for the common gene mutations. In families in which the diseasecausing mutations are known, molecular testing can be used to accurately identify carriers.
DNA analysis for the following seven Gaucher disease mutations are performed for the Ashkenazic Jewish population. The
frequency of these mutations is given below.
GAUCHER’S DISEASE MUTATION ANALYSIS
Mutation
1226G
(N370S)
84GG
1448C
Frequency (Non-jewish)
75% (25% non-jewish)
13%
(L444P)
5% (40% non-jewish)
1604A
(R496H)
1%
1297T
(V394L)
rare
1504T
(R463C)
(Non-jewish)
IVS2+1
3%
The mutations listed above account for ~97% of the Gaucher disease mutations in the Jewish population.(Beutler et. al. Am.
J. Hum. Genet. (1993) 52:85-88). DNA analysis for these mutations is based on specific PCR amplification, mismatched PCR
followed by restriction digestion and/or allele specific oligonucleotide hybridization.
Mutation 1226G is the most common cause of Gaucher disease in Jewish patients and is associated with a mild late-onset
clinical phenotype. Only about one third individuals with 1226G/1226G genotype manifest symptoms. Patients who are
compound heterozygotes for mutation 1226G and 84GG have a more severe clinical disorder than those who are homozygous
for the 1226G mutation. The median age of first symptoms in 1226G/1226G is 30.5 yrs. compared to 6 years for
1226G/84GG. No patients homozygous for for 84GG mutation have been reported probably indicating this genotype would be
lethal before birth. Mutation 1448C is associated with a more severe disease as compared to 1226G. A 1448C/1448C
genotype predicts a severe form of neuropathic Gaucher disease as does the IVS2 muatation.
Beutler's analysis of mutation frequency of all Gaucher mutations is about 0.031 in the Ashkenazi Jewish population, and the
frequency of the 1226G mutation to be about 0.028 and that of the 84GG mutation 0.0028. Therefore the frequency of all
alleles other than 1226G, 84GG and 1448C would be 3.3% of the total, or 1x10-3. A Jewish couple who is negative for the
1226G, 84GG and 1448C will have only 1 in about 1,000,000 risk of having a affected. Whereas, if one partner has one of the
three mutations and the other none of these three, the risk will be 1:1000. The estimated carrier frequency for Gaucher
disease in Ashkenazic Jewish individual is 1/11.
Molecular analysis
DNA analysis for Gaucher's disease mutations is accomplished by specific PCR amplification, mismatched PCR (discussed
below), followed by restriction endonuclease digestion and/or ASO hybridization. We previously discussed PCR followed by
restriction endonuclease digestion and ASO. Here we will discuss a new method of mutation detection, mismatched PCR.
Not all mutations result in the gain or loss of a restriction site. Such mutations therefore cannot be analyzed by
PCR/restriction endonuclease method. ASO requires the use of radioactivity and thorough optimization. The mismatched PCR
method was introduced by Beutler et al27 to overcome these difficulties. In this method, one of the primers for PCR is
constructed in a way that the 3' end of the DNA strand adjacent to the site of the mutation and the internal sequence of the
primer is altered so that a restriction endonuclease site will either be gained or lost once the PCR product is amplified.
The example given in Fig 3A is for Gaucher's disease mutation 1226G (also known as N370S). In this mutation, an A is
changed to a G at position 1226, leading to the substitution of the amino acid serine for asparagine. This mutation does not
create or abolish a site for any known/commercially available restriction endonuclease.
- 95 -
One of the PCR primers is constructed with a mismatch, as shown in Fig 3B. Primers with internal mismatches will hybridize to
target sequences at optimized conditions, and elongation of this primer with a normal template wil1 result in the addition of
an A residue; in the mutant template, a G residue will be added. The use of the mismatched primer in concert with a 1226G
mutant template creates a new Xho I restriction endonuclease site (Table 6). Digestion of PCR products from normal and
1226G mutant templates is fol1owed by electrophoretic separation. The result wil1 be two fragments for the mutant product
(it will be cleaved), whereas the normal product remains uncleaved, resulting in visualization of a single, higher molecular
weight fragment. This technique is reliable, and it is performed a fashion very similar to PCR, fol1owed by restriction
endonuclease digestion. This mismatched PCR method may also be used for the 84GG Gaucher mutation.27
Detection Methods
The choice of using methods for mutation analysis is restricted by our knowledge of the existence of a mutation and its type
and in parallel to the development of safe and sensitive new detection methods. Gene Link’s primary focus is in the
development of facile non-radioactive detection methods. This product profile of Gene Link’s current gene detection product
line spotlights chemiluminescent detection based products as well as conventional radioactive based methods. The gene
detection systems can be divided into five broad groups based on detection methods:
Genemer™: Genemer™ is comprised of a primer pair for PCR amplification of amplification of the fragment of interest and
visualization of the product by gel electrophoresis and ethidium bromide staining. This product contains one tube containing
10 nmol of forward and reverse lyophilized primer. The quantity supplied is sufficient for 400 regular 50 µl PCR reactions. The
10 nmol of primer when dissolved in 50µl water will give a solution of 200 µMolar i.e. 200 pmol/µl.
Genemer™ Kit: The Genemer™ kit is a complete easy-to-use kit for reliable genotyping of a gene fragment. This line of
products is PCR based. The product includes a specific primer pair for gene or mutation specific amplification, optimized
buffers and dNTPs and in most cases, control DNA. Kit includes sufficient reagents for 100 detections.
Genemer™ Control DNA: These are cloned fragment of the mutation region of a particular gene for use with gene or
mutation specific Genemer™ products. These control DNAs are ideal genotyping templates for optimizing and performing
control amplification with unknown DNA. One tube is supplied containing 500 ng of lyophilized DNA segment of the specified
region. The quantity supplied is sufficient for 1000 regular 50 µl PCR reactions.
Results and Interpretation
1226G
84GG
1448C
IVS2+1
1604A
1297T
1504T
Mutation
(N370S)
PCR Product
105
75
102
357
170
69
775
(L444P)
(R496H)
(V394L)
(R463C)
Normal
105
75
102
141+117+99
170
69
615 +150
Homo
89+16
57+18
57+45
240+117
128 +42
49+22
775
Carrier
105+89+16
75+57+18
102+57+45
240+141+117+99
170 +128 +42
69 +47 +22
775+615+160
*The polymerase chain reaction (PCR) process is covered by patents owned by Hoffmann-La Roche. A license to perform is automatically granted by the use of authorized reagents.
References:
1. Beutler et.al. Clin. Chim. Acta (1990) 194:161-166.
2. Beutler et. al. PNAS (1991) 88:10544-10547.
3. Zimran...Beutler Lancet (1989) 349-352.
4. Beutler Science (1992) 256:794-799.
5. Horowitz, M. and Zimran,A Human Mutation (1994) 3:1-11
Ordering Information
Product
Size
Catalog No.
Price, $
Gaucher 1226G (N370S) Genemer™
10 nmols
40-2047-12
100.00
Gaucher 84GG Genemer™
10 nmols
40-2047-13
100.00
Gaucher 1448C (L444P) Genemer™
10 nmols
40-2047-14
100.00
Gaucher IVS2+1 Genemer™
10 nmols
40-2047-15
100.00
Gaucher 1604A (R496H) Genemer™
10 nmols
40-2047-16
100.00
Gaucher 1297T (V394L) Genemer™
10 nmols
40-2047-17
100.00
Gaucher 1504T (R463C) Genemer™
10 nmols
40-2047-18
100.00
- 96 -
Gene Detection Systems
By Product Line
- 97 -
- 98 -
GeneProber™
The GeneProber™ product line is based on the chemiluminescent Southern blot detection method. Gene Link’s non-radioactive
detection systems for genotyping of triple repeat disorders are rapid, reliable and as sensitive as the 32P labeled southern
blots. No more decayed probes and radioactive exposure. Kits are available for reliable genotyping of the following triple
repeat mutation group disorders.
GeneProber™ Unlabeled Probes
Product
Fragile X GeneProber™ GLFX1 Probe unlabeled
Fragile X CGG triple repeat spanning region unlabeled probe for radioactive labeling
and Southern blot detection. Suitable for random primer labeling.
Size
Catalog No.
Price, $
500 ng
40-2004-40
350.00
500 ng
40-2025-40
350.00
500 ng
40-2026-40
350.00
500 ng
40-2026-39
350.00
500 ng
40-2026-38
350.00
500 ng
40-2027-40
350.00
500 ng
40-2032-40
350.00
Huntington Disease GeneProber™ GLHD Probe unlabeled
Huntington Disease CAG triple repeat spanning region unlabeled probe for
radioactive labeling and Southern blot detection. Suitable for random primer
labeling.
Myotonic Dystrophy GeneProber™ GLDM1 Probe unlabeled.
Myotonic dystrophy CTG triple repeat spanning region unlabeled probe for
radioactive labeling and Southern blot detection of Bam HI digested DNA. Suitable
for random primer labeling.
Myotonic Dystrophy GeneProber™ GLDM2 Probe unlabeled.
Myotonic dystrophy CTG triple repeat spanning region unlabeled probe for
radioactive labeling and Southern blot detection of Pst I digested DNA. Suitable for
random primer labeling.
Myotonic Dystrophy GeneProber™ GLDM3 Probe unlabeled.
Myotonic dystrophy CTG triple repeat spanning region unlabeled probe for
radioactive labeling and Southern blot detection. Suitable for random primer
labeling.
Friedreich Ataxia GeneProber™ FRDA-GL3 Probe unlabeled
Friedreich Ataxia GAA triple repeat spanning region unlabeled probe for radioactive
labeling and Southern blot detection. Suitable for random primer labeling.
Kennedy Disease (SBMA) GeneProber™ GLSBMA Probe unlabeled
Kennedy Disease CAG triple repeat spanning region unlabeled probe for radioactive
labeling and Southern blot detection. Suitable for random primer labeling.
- 99 -
GeneProber™ Digoxigenin Labeled Probes
Product
Fragile X GeneProber™ GLFXDig1 Probe Digoxigenin labeled
Fragile X CGG triple repeat spanning region digoxigenin labeled probe for nonradioactive Southern blot detection.
Huntington Disease GeneProber™ GLHD Probe Digoxigenin labeled
Huntington Disease CAG repeat spanning region digoxigenin labeled probe for nonradioactive detection Southern blot.
Myotonic Dystrophy GeneProber™ GLDMDig2 Probe Digoxigenin labeled.
Myotonic dystrophy CTG triple repeat spanning region digoxigenin labeled probe for
Southern blot non-radioactive detection of Pst I digested DNA.
Friedreich Ataxia GeneProber™ GLFRDA Probe Digoxigenin labeled
Friedreich Ataxia GAA triple repeat spanning region digoxigenin labeled probe for
Southern blot non-radioactive detection.
Kennedy Disease (SBMA) GeneProber™ GLSBMA Probe Digoxigenin
labeled
Kennedy Disease CAG triple repeat spanning region digoxigenin labeled probe for
non-radioactive Southern blot detection.
Size
Catalog No.
Price, $
110 µl
40-2004-41
400.00
110 µl
40-2025-41
400.00
110 µl
40-2026-41
400.00
110 µl
40-2027-41
400.00
110 µl
40-2032-41
400.00
- 100 -
PCRProber™ Gene Detection Kits
Gene Link’s PCRProber™ Kit is based on PCR amplification
followed by Southern blot chemiluminescent detection using
an Alkaline Phosphatase labeled oligonucleotide probe. This
kit is a safe and sensitive alternate to radioactive-based
detection methods. The amplified products are resolved on a
sequencing polyacrylamide gel, and then blotted and
processed for chemiluminescent detection.
The PCRProber™ Kit is simple and robust for routine triple
repeat detection of greater than 100 repeats of all triple
repeat disorders listed, except Fragile X. The CGG repeat in
Fragile X can be detected up to ~50 repeats.
It is strongly recommended that the genotyping be followed
up by using Southern blot detection methods when two
alleles are not clearly discernable. Quantity supplied is 1 kit
[100 rxns].
PCRProber ™ Kits
Product
Fragile X PCRProber™ Kit (spanning triple repeat region)
Kit for performing PCR amplification and chemiluminescent based detection.
Huntington Disease PCRProber ™ Kit (spanning triple repeat region)
Kit for performing PCR amplification and chemiluminescent based detection.
Myotonic PCRProber™ Kit (spanning triple repeat region)
Kit for performing PCR amplification and chemiluminescent based detection.
Friedreich Ataxia PCRProber ™ Kit (spanning triple repeat region)
Kit for performing PCR amplification and chemiluminescent based detection.
CAG repeat PCRProber™ Kit (spanning triple repeat region)
Kit for performing PCR amplification and chemiluminescent based detection
for all CAG triple repeat disorders.
Size
Catalog No.
Price, $
1 kit
40-2004-32
650.00
1 kit
40-2025-32
650.00
1 kit
40-2026-32
650.00
1 kit
40-2027-32
650.00
1 kit
40-20XX-32
650.00
PCRProber ™ Alkaline Phosphatase Labeled Probes
Product
Fragile X PCRProber™ AP labeled probe
Alkaline phosphatase labeled probe
Huntington Disease PCRProber™ AP labeled probe
Alkaline phosphatase labeled probe
Myotonic Dystrophy PCRProber™ AP labeled probe
Alkaline phosphatase labeled probe
Friedreich Ataxia PCRProber™ AP labeled probe
Alkaline phosphatase labeled probe
CAG repeat PCRProber™ AP labeled probe
Alkaline phosphatase labeled probe
Size
Catalog No.
Price, $
12 µl
40-2004-31
400.00
12 µl
40-2025-31
400.00
12 µl
40-2026-31
400.00
12 µl
40-2027-31
400.00
12 µl
40-20XX-31
400.00
- 101 -
Genemer™
The Genemer™ product line is PCR based. The product includes a specific primer pair for gene or mutation specific
amplification. Easy to use, reliable genotyping kits are available with control DNA. Genemer products are available for the
gene fragment and disorder listed. Specialized optimized conditions may be required for certain triple repeat disorder
amplifications. Gene Link recommends the Genemer™ kits for researchers who have not established their own optimized
amplification conditions. The Genemer™ kits contain optimized buffers and primers.
This product contains one tube containing 10 nmols of forward and reverse lyophilized primer. The quantity supplied is
sufficient for 400 regular 50µl PCR reaction. The 10 nmols of primer when dissolved in 50µl water will give a solution of 200
µMolar i.e. 200 pmole/µl.
Genemer™
Product
Size
Catalog No.
Sickle Cell Genemer™
10 nmols
40-2001-10
RhD (RhD gene exon 10 specific) Genemer™
10 nmols
40-2002-10
Rh EeCc (Rh Ee and Cc exon 7 specific) Genemer™
10 nmols
40-2003-10
Fragile X (spanning CGG triple repeat region) Genemer™
10 nmols
40-2004-10
SRY (sex determining region on Y) Genemer™
10 nmols
40-2020-10
X alphoid repeat Genemer™
10 nmols
40-2021-10
Y alphoid repeat Genemer™
10 nmols
40-2022-10
Huntington Disease (spanning CAG triple repeat region) Genemer™
10 nmols
40-2025-10
Myotonic Dystrophy (spanning CTG triple repeat region) Genemer™
10 nmols
40-2026-10
Friedreich’s Ataxia (spanning GAA triple repeat region) Genemer™
10 nmols
40-2027-10
Cystic Fibrosis (various mutations) Genemer™
10 nmols
40-2029-XX
Kennedy Disease Genemer™
10 nmols
40-2032-10
Gaucher (various mutations) Genemer Kit
10 nmols
40-2047-XX
*Please visit www.genelink.com for other Genemer™ not listed here
Price, $
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
- 102 -
Genemer™ Kits
The Genemer™ kit is a complete easy-to-use kit for reliable
genotyping of a gene fragment. This line of products is PCR
based. The product includes a specific primer pair for gene or
mutation specific amplification, optimized buffers and dNTPs
and in most cases, control DNA. These kits contain
specialized and optimized conditions that are required for
amplification of large repeats in certain triple repeat disorder
amplifications. Gene Link recommends these Genemer™ kits
for researchers who have not established their own optimized
amplification conditions.
Gene Link has developed a series of PCR systems capable of
routinely amplifying greater than 150 CAG repeats for
Huntington and other CAG triple repeat disorders. The PCR
system is thus capable to detect all CAG triple repeat
disorders. This system uses regular Taq polymerase.
Amplified fragments are resolved by convenient agarose gel
electrophoresis and ethidium bromide staining.
It is to be emphasized that accurate size determination
should be done on polyacrylamide sequencing gel or a
fluorescent
genetic
analyzer
using
GeneProber™,
PCRProber™, or GScan™ kits. Genemer™ kits are available
for the gene fragment and disorders listed below. Kit includes
sufficient reagents for 100 detections.
Genemer™ Kits
Product
Size
Catalog No.
Price, $
Fragile X (spanning CGG triple repeat region) available as GeneProber™, PCRProber™, or GScan™ kits
Huntington Disease (spanning CAG triple repeat region) Genemer Kit
1 Kit
40-2025-11
250.00
Myotonic Dystrophy (spanning CTG triple repeat region) Genemer Kit
1 Kit
40-2026-11
250.00
Friedreich’s Ataxia (spanning GAA triple repeat region) Genemer Kit
1 Kit
40-2027-11
250.00
Kennedy Disease (SBMA) Genemer Kit
1 Kit
40-2032-11
250.00
Sickle Cell Genemer Kit
1 Kit
40-2001-11
200.00
RhD (RhD gene exon 10 specific) Genemer Kit
1 Kit
40-2002-11
200.00
Rh EeCc (Rh Ee and Cc exon 7 specific) Genemer Kit
1 Kit
40-2003-11
200.00
SRY (sex determining region on Y) Genemer Kit
1 Kit
40-2020-11
200.00
X alphoid repeat Genemer Kit
1 Kit
40-2021-11
200.00
Y alphoid repeat Genemer Kit
1 Kit
40-2022-11
200.00
Cystic Fibrosis (various mutations) Genemer Kit
1 Kit
40-2029-XXK
200.00
Gaucher (various mutations) Genemer Kit
1 Kit
40-2047-XXK
200.00
*Please visit www.genelink.com for other Genemer™ Kits not listed here
- 103 -
Genemer™ Radioactive Detection Kits
Gene Link strongly recommends the use of non-radioactive gene detection systems.
Consider switching to Gene Link’s product line of non-radioactive detection systems.
Genemer™ kits are also available for conventional radioactive-based detection methods. A Radioactive component is not
present in these kits. Genemer™ kits are complete easy-to-use kits for reliable genotyping of a gene fragment. This line of
products is PCR based. The product includes a specific primer pair for gene or mutation specific amplification, optimized
buffers and dNTPs and in most cases control DNA. These kits contain specialized and optimized conditions that are required
for amplification of large repeats in certain triple repeat disorder amplifications.
Gene Link has developed a series of PCR systems capable of routinely amplifying greater than 150 CAG repeats for
Huntington and other CAG triple repeat disorders. The PCR system is thus capable to detect all CAG triple repeat disorders.
This system uses regular Taq polymerase.
It is to be emphasized that accurate size determination should be done on polyacrylamide sequencing gel. Genemer™
radioactive use kits are available for the gene fragment and disorder listed below. Kit includes sufficient reagents for 100
detections.
Genemer™ Radioactive Detection Kits
Product
Fragile X Genemer™ Kit for Radioactive Detection
Kit for amplification and radioactive detection of Fragile X CGG triple
repeat region amplified PCR products using 35S or 32P.
Huntington Disease Genemer™ Kit for Radioactive Detection
Kit for amplification and radioactive detection of Huntington Disease
CAG triple repeat region amplified PCR products using 35S or 32P.
Myotonic Dystrophy Genemer™ Kit for Radioactive Detection
Kit for amplification and radioactive detection of Myotonic Dystrophy
CTG triple repeat region amplified PCR products using 35S or 32P.
Friedreich’s Ataxia Genemer™ Kit for Radioactive Detection
Kit for amplification and radioactive detection of Friedreich’s Ataxia
GAA triple repeat region amplified PCR products using 35S or 32P.
Kennedy Disease Genemer™ Kit for Radioactive Detection
Kit for amplification and radioactive detection of Kennedy Disease
CAG triple repeat region amplified PCR products using 35S or 32P.
Size
Catalog No.
Price, $
1 Kit
40-2004-20
350.00
1 Kit
40-2025-20
350.00
1 Kit
40-2026-20
350.00
1 Kit
40-2027-20
350.00
1 Kit
40-2032-20
350.00
Please visit www.genelink.com for other Genemer™ Kits not listed here
- 104 -
GScan™ Gene Detection Kits
Genotyping using this kit requires use of the appropriate fluorescent genetic analyzer instrument(s) and software capable of
detection of fluorescently labeled fragments of varying lengths. These kist has been optimized for an ABI310 genetic
analyzer.
Gene Link’s GScan™ gene detection kits are safe, convenient
and sensitive, and afford automated compilation of data. The
kits contain optimized PCR amplification reagents and a wide
selection of fluorescent-labeled primers for genotyping after
PCR using fluorescent genetic analyzer instrument(s).
Included in these kits are ready to run control samples of
various repeats of the triple repeat disorder kit. These control
samples are for calibration with the molecular weight
markers for accurate size determination of the amplified
fragments.
The GScan kits are simple and robust for routine triplerepeat detection of greater than 100 repeats of all triple
repeat disorders listed, except Fragile X. The CGG repeat in
Fragile X can be detected up to ~50 repeats.
It is strongly recommended that the genotyping be followed
up by using Southern blot detection methods when two
alleles are not clearly discernable. Kit includes sufficient
reagents for 100 detections.
GScan™ Gene Detection Kits
Product
Size
Fragile X GScan TET Kit
1 Kit
Fragile X GScan HEX Kit
1 Kit
Fragile X GScan 6-FAM Kit
1 Kit
Fragile X GScan Cy3 Kit
1 Kit
Fragile X GScan Cy5 Kit
1 Kit
Huntington Disease GScan TET Kit
1 Kit
Huntington Disease GScan HEX Kit
1 Kit
Huntington Disease GScan 6-FAM Kit
1 Kit
Huntington Disease GScan Cy3 Kit
1 Kit
Huntington Disease GScan Cy5 Kit
1 Kit
Myotonic Dystrophy GScan TET Kit
1 Kit
Myotonic Dystrophy GScan HEX Kit
1 Kit
Myotonic Dystrophy GScan 6-FAM Kit
1 Kit
Myotonic Dystrophy GScan Cy3 Kit
1 Kit
Myotonic Dystrophy GScan Cy5 Kit
1 Kit
Friedreich’s Ataxia GScan TET Kit
1 Kit
Friedreich’s Ataxia GScan HEX Kit
1 Kit
Friedreich’s Ataxia GScan 6-FAM Kit
1 Kit
Friedreich’s Ataxia GScan Cy3 Kit
1 Kit
Friedreich’s Ataxia GScan Cy5 Kit
1 Kit
Kennedy Disease GScan TET Kit
1 Kit
Kennedy Disease GScan HEX Kit
1 Kit
Kennedy Disease GScan 6-FAM Kit
1 Kit
Kennedy Disease GScan Cy3 Kit
1 Kit
Kennedy Disease GScan Cy5 Kit
1 Kit
Please visit www.genelink.com for other GScan™ Kits
Catalog No.
40-2004-15TT
40-2004-15HX
40-2004-15FM
40-2004-15C3
40-2004-15C5
40-2025-15TT
40-2025-15HX
40-2025-15FM
40-2025-15C3
40-2025-15C5
40-2026-15TT
40-2026-15HX
40-2026-15FM
40-2026-15C3
40-2026-15C5
40-2027-15TT
40-2027-15HX
40-2027-15FM
40-2027-15C3
40-2027-15C5
40-2032-15TT
40-2032-15HX
40-2032-15FM
40-2032-15C3
40-2032-15C5
not listed here.
Price, $
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
650.00
- 105 -
GScan™ Genemer Control DNA
PCR amplified HEX labeled fragment of the mutation region of a particular gene for use with gene or mutation specific
Genemer™. These control DNAs are ideal genotyping templates for optimizing and performing control amplification with
unknown DNA. The size of the triple repeats has been determined by sequencing and gel electrophoresis. The stability of size
repeats upon cloning and amplification has NOT been determined. Thus, the size should be considered approximate and there
is no claim for each fragment to contain the exact number of triple repeats. These control DNAs are sold with the expressed
condition that these NOT be used for exact triple repeat size determination of DNA of unknown genotype. These control DNA
should be used for calibration and determining the performance of specific Genemer™ kits.
Product
Size
Catalog No.
GLFX ~16 CGG repeat GScan Genemer Control DNA; HEX labeled
25 µl
40-2004-01HX
GLFX ~29 CGG repeat GScan Genemer Control DNA; HEX labeled
25 µl
40-2004-02HX
GLFX ~40 CGG repeat GScan Genemer Control DNA; HEX labeled
25 µl
40-2004-03HX
GLHD 7 ~CAG repeat GScan Genemer Control DNA; HEX labeled
25 µl
40-2025-05HX
GLHD 18 ~CAG repeat GScan Genemer Control DNA; HEX labeled
25 µl
40-2025-01HX
GLHD 31 ~CAG repeat GScan Genemer Control DNA; HEX labeled
25 µl
40-2025-07HX
GLHD 34 ~CAG repeat GScan Genemer Control DNA; HEX labeled
25 µl
40-2025-02HX
GLHD 37 ~CAG repeat GScan Genemer Control DNA; HEX labeled
25 µl
40-2025-08HX
GLHD 44 ~CAG repeat GScan Genemer Control DNA
25 µl
40-2025-03HX
GLHD 49 ~CAG repeat GScan Genemer Control DNA
25 µl
40-2025-09HX
GLHD 89 ~CAG repeat GScan Genemer Control DNA
25 µl
40-2025-04HX
GLHD 116 ~CAG repeat GScan Genemer Control DNA
25 µl
40-2025-06HX
GLHD 134 ~CAG repeat GScan Genemer Control DNA
25 µl
40-2025-61HX
GLHD 182 ~CAG repeat GScan Genemer Control DNA
25 µl
40-2025-62HX
GLDM 12 ~CTG repeat GScan Genemer™ Control DNA
25 µl
40-2026-01HX
GLDM 45 ~CTG repeat GScan Genemer™ Control DNA
25 µl
40-2026-02HX
GLDM 93 ~CTG repeat GScan Genemer™ Control DNA
25 µl
40-2026-03HX
GLDM 129 ~CTG repeat GScan Genemer™ Control DNA
25 µl
40-2026-04HX
GLDM 182 ~CTG repeat GScan Genemer™ Control DNA
25 µl
40-2026-05HX
GLFRDA ~64 GAA repeat GScan Genemer Control DNA
25 µl
40-2027-01HX
GLFRDA ~102 GAA repeat GScan Genemer Control DNA
25 µl
40-2027-02HX
GLFRDA ~110 GAA repeat GScan Genemer Control DNA
25 µl
40-2027-03HX
GLFRDA ~125 GAA repeat GScan Genemer Control DNA
25 µl
40-2027-04HX
GLFRDA ~9 GAA repeat GScan Genemer Control DNA
25 µl
40-2027-05HX
Kennedy (SBMA) ~23 CAG repeat GScan Genemer Control DNA
25 µl
40-2032-01HX
Please visit www.genelink.com for other GScan Genemer™ Controls not listed here
Price, $
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
Huntington’s Disease Control DNA with 134 CAG repeats
- 106 -
Genemer™ Control DNA
These are cloned fragment of the mutation region of a particular gene for use with gene or mutation specific Genemer™
products. These control DNAs are ideal genotyping templates for optimizing and performing control amplification with
unknown DNA. The size of the triple repeats has been determined by sequencing and gel electrophoresis. The stability of size
repeats upon cloning and amplification has NOT been determined. Thus, the size should be considered approximate and there
is no claim for each fragment to contain the exact number of triple repeats. These control DNAs are sold with the expressed
condition that these NOT be used for exact triple repeat size determination of DNA of unknown genotype. The control DNA
should be used for determining the performance of specific Genemer™ and PCRProber™ Gene Link products.
Product
Size
Catalog No.
GLFX ~16 CGG repeat Genemer Control DNA
500 ng
40-2004-01
GLFX ~29 CGG repeat Genemer Control DNA
500 ng
40-2004-02
GLFX ~40 CGG repeat Genemer Control DNA
500 ng
40-2004-03
GLFX ~60 CGG repeat Genemer Control DNA
500 ng
40-2004-04
GLFX ~90 CGG repeat Genemer Control DNA
500 ng
40-2004-05
GLHD ~7 CAG repeat Genemer Control DNA
500 ng
40-2025-05
GLHD ~18 CAG repeat Genemer Control DNA
500 ng
40-2025-01
GLHD ~31 CAG repeat Genemer Control DNA
500 ng
40-2025-07
GLHD ~34 CAG repeat Genemer Control DNA
500 ng
40-2025-02
GLHD ~37 CAG repeat Genemer Control DNA
500 ng
40-2025-08
GLHD ~44 CAG repeat Genemer Control DNA
500 ng
40-2025-03
GLHD ~49 CAG repeat Genemer Control DNA
500 ng
40-2025-09
GLHD ~89 CAG repeat Genemer Control DNA
500 ng
40-2025-04
GLHD ~116 CAG repeat Genemer Control DNA
500 ng
40-2025-06
GLHD ~134 CAG repeat Genemer Control DNA
500 ng
40-2025-61
GLHD ~182 CAG repeat Genemer Control DNA
500 ng
40-2025-62
GLDM ~12 CTG repeat Genemer™ Control DNA
500 ng
40-2026-01
GLDM ~45 CTG repeat Genemer™ Control DNA
500 ng
40-2026-02
GLDM ~93 CTG repeat Genemer™ Control DNA
500 ng
40-2026-03
GLDM ~129 CTG repeat Genemer™ Control DNA
500 ng
40-2026-04
GLDM ~182 CTG repeat Genemer™ Control DNA
500 ng
40-2026-05
GLFRDA ~64 GAA repeat Genemer Control DNA
500 ng
40-2027-01
GLFRDA ~102 GAA repeat Genemer Control DNA
500 ng
40-2027-02
GLFRDA ~110 GAA repeat Genemer Control DNA
500 ng
40-2027-03
GLFRDA ~125 GAA repeat Genemer Control DNA
500 ng
40-2027-04
GLFRDA ~9 GAA repeat Genemer Control DNA
500 ng
40-2027-05
Kennedy Disease (SBMA) ~23 CAG repeat Genemer Control DNA
500 ng
40-2032-01
Sickle Cell HbA Genemer Control DNA
500 ng
40-2001-01
Sickle Cell HbS Genemer Control DNA
500 ng
40-2001-02
Sickle Cell HbC Genemer Control DNA
500 ng
40-2001-03
RhD (RhD gene exon 10 specific) Genemer Control DNA
500 ng
40-2002-01
Rh EeCc (Rh Ee and Cc exon 7 specific) Genemer Control DNA
500 ng
40-2003-01
SRY (sex determining region on Y) Genemer Control DNA
500 ng
40-2020-01
X alphoid repeat Genemer Control DNA
500 ng
40-2021-01
Y alphoid repeat Genemer Control DNA
500 ng
40-2022-01
Please visit www.genelink.com for other Genemer™ Control DNA not listed here.
Hb-A: …TCCTGAGGAG…
Hb-S: …TCCTGTGGAG…
Price, $
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
175.00
115.00
115.00
115.00
115.00
115.00
115.00
115.00
115.00
Hb-C: …TCCTAAGGAG…
*The polymerase chain reaction (PCR) process is covered by patents owned by Hoffmann-La Roche. A license to perform is automatically
granted by the use of authorized reagents.
**Boehringer Mannheim/Roche holds exclusive rights to digoxigenin labeling. Digoxigenin oligo labeling is offered under license from Roche.
Extensive digoxigenin labeling techniques and detection methods are available from Roche
Prices subject to change without notice.
All Gene Link products are for research use only.
- 107 -
- 108 -
GENETIC TOOLS AND REAGENTS
- 109 -
- 110 -
RT-PCRmer™
RT-PCRmerTM are primer pairs for specific amplification of cDNA. ß-actin is ubiquitously expressed and serves as a positive
control for northern and other expression studies. ß-actin RT-PCRmerTM is generally used as controls for measuring cDNA
synthesis efficiency by reverse transcription and as controls for mRNA (cDNA) quantitative expression studies. ß-actin RTPCRmerTM are supplied as a lyophilized powder in aliquots of 10nmoles. The 10nmoles of primer when dissolved in 500µl
sterile water or TE will give a solution of 20µMolar i.e. 20pmoles/µl. The quantity supplied is sufficient for at least 400 regular
25µl PCR reaction* for ethidium bromide stained visualization. This primer set will amplify a fragment of 289 bp from human
and rat cDNA. The fragments can be distinguished from rat or human source by digestion with Pvu II; the rat amplified 289
bp fragment is digested to give a 132 and 157 fragments whereas the human amplified fragment is not digested due to the
absence of the Pvu II (1).
The product is supplied as a lyophilized powder, after reconstitution store at -20oC. Oligo purity is greater than 98% as
determined by denaturing polyacrylamide gel electrophoresis.
RT-PCRmer™
Product
Catalog No.
Quantity
Price $
RT-PCRmer; Human beta actin
RT-PCRmer; Rat beta actin
40-1001-10
40-1002-10
10 nmols
10 nmols
100.00
RT-PCRmer; Mouse beta actin
40-1003-10
40-1004-10
10 nmols
100.00
10 nmols
100.00
40-1005-10
40-1002-00
10 nmols
100.00
10 nmols
100.00
RT-PCRmer; beta 2 microglobulin
RT-PCRmer; GAPDH H/M/R
RT-PCRmer; Beta actin control PCR mix (human &
t)
100.00
*Please see our First Strand cDNA section for related products.
OligoProber™
OligoProber™ are specific oligonucleotide probes for hybridization to its cognate species. These are specially
suited for use in conjunction with RT-PCRmers™, as the complementary sequence lies in the amplified
sequence. The OligoProber™ can also be used for all northern blots. OligoProber™ are available for use as
hybridization probes with either 5’OH for 32P labeling or with 3’ biotin for non-radioactive detection. The
OligoProber™ is supplied as a lyophilized powder in aliquots of 2nmoles. The 2 nmoles of primer when dissolved
in 100µl sterile water or TE will give a solution of 20 µMolar i.e. 20 pmoles/µl. Oligo purity is greater than 98%
as determined by denaturing polyacrylamide gel electrophoresis.
Reference
1. du Breuil, R. M., Patel, J.M. and Mendelow, B.V. (1993) PCR methods and applications. 3:57-59.
OligoProber™
Catalog No.
Quantity
Price $
TM
Product
40-1101-02
2 nmols
55.00
TM
40-1102-02
2 nmols
55.00
TM
40-1103-02
2 nmols
55.00
40-1105-02
2 nmols
55.00
OligoProber ; Biotinylated Human beta actin
40-1111-02
2 nmols
150.00
OligoProberTM; Biotinylated Rat beta actin
40-1112-02
2 nmols
150.00
40-1113-02
2 nmols
OligoProber ; Human beta actin
OligoProber ; Rat beta actin
OligoProber ; Mouse beta actin
OligoProberTM; GAPDH H/M/R
TM
150.00
OligoProber ; Biotinylated Mouse beta actin
TM
OligoProber ; Biotinylated GAPDH H/M/R
40-1115-02
2 nmols
150.00
*The polymerase chain reaction (PCR) process is covered by patents owned by Hoffmann-La Roche. A license to perform is
automatically granted by the use of authorized reagents.
TM
- 111 -
Omni-Ladder™ Unlabeled DNA Molecular Weight Markers
Omni-Marker™ Universal and Low are unlabeled DNA markers containing a blend of fragments ranging from
50 base pairs to 10 kb. The universal contains fragments of the following sizes; 10 kb, 8 kb, 6 kb, 4 kb, 3 kb,2
kb, 1.55, 1.4 kb, 1 kb, 750 bp, 500 bp, 400 bp kb, all the bands and “low” version contains fragments from 50
bp to 2kb. The low Omni-Marker™ is ideal for routine PCR gels. A loading of 5 µl is sufficient per lane.
Omni-Marker™ Universal unlabeled
Omni-Marker™ Low unlabeled
Fragment Size
10 kb
8 kb
6 kb
4 kb
3 kb
2 kb
1.55 kb
1.40 kb
1.00 kb
750 bp
500 bp
400 bp
300 bp
200 bp
100 bp
50 bp
Fragment Size
2 kb
1.55 kb
1.40 kb
1.00 kb
750 bp
500 bp
400 bp
300 bp
200 bp
100 bp
50 bp
Approx. conc.
30 ng
30 ng
45 ng
60 ng
85 ng
150 ng
100 ng
100 ng
120 ng
30 ng
60 ng
20 ng
40 ng
30 ng
20 ng
15 ng
Approx. conc.
150 ng
100 ng
100 ng
120 ng
30 ng
60 ng
20 ng
40 ng
30 ng
20 ng
15 ng
The gel picture shows the fragments obtained by electrophoresing in 1.5% agarose gel. The low and universal
Omni-Markers are provided premixed with or without dye. The marker and dye both contain 0.02% sodium
azide.
Molecular Weight Markers
Product
Catalog No.
Size
Price $
Omni-Marker™ Universal unlabeled
40-3005-01
100 µl
15.00
Omni-Marker™ Universal unlabeled
40-3005-05
500 µl
50.00
Omni-Marker™ Universal unlabeled
40-3005-10
1 ml
90.00
Omni-Marker™ Low unlabeled
40-3006-01
100 µl
15.00
Omni-Marker™ Low unlabeled
40-3006-05
500 µl
50.00
Omni-Marker™ Low unlabeled
40-3006-10
1 ml
90.00
* Normal recommended loading per lane is 5 µl . Shipped at room temperature. Store at -20oC
112
Omni-Ladder™ Labeled DNA Molecular Weight Markers
Omni-Marker™ dye labeled, Alkaline Phosphatase, biotin and digoxigenin labeled MW Markers are also available.
Omni-Marker™ Dye Labeled MW Markers
Product
Catalog No.
Size*
Price $
Omni-Marker™ GScan-1 Tamra labeled 50 bp-1kb
40-3061-01
100 µl
95.00
Omni-Marker™ GScan-1 Tamra labeled 50 bp-1kb
40-3061-05
500 µl
395.00
Omni-Marker™ GScan-2 Tamra labeled 50 bp- 600 bp
40-3062-01
100 µl
95.00
Omni-Marker™ GScan-2 Tamra labeled 50 bp- 600 bp
40-3062-05
500 µl
395.00
Omni-Marker™ GScan-1 Tet labeled 50 bp-1kb
40-3071-01
100 µl
95.00
Omni-Marker™ GScan-1 Tet labeled 50 bp-1kb
40-3071-05
500 µl
395.00
Omni-Marker™ GScan-2 Tet labeled 50 bp- 600 bp
40-3072-01
100 µl
95.00
Omni-Marker™ GScan-2 Tet labeled 50 bp- 600 bp
40-3072-05
500 µl
395.00
o
*A loading of 0.5µl is suggested. Shipped at room temperature. Store at -20 C
Loading Buffers
Gene Link also supplies loading buffers. We recommend Orange G for very low molecular weight DNA, as it
usually runs around the 20-30bp range on 1% agarose. Bromophenol Blue /Xylene Cyanol DNA loading buffer
is better for DNA of larger molecular weight.
Loading Buffers
Product
Catalog No.
Size
Price $
5X BPB/XC non-denaturing loading buffer
40-3002-01
100 µl
5.00
5X BPB/XC non-denaturing loading buffer
40-3002-10
1ml
10.00
5X Orange G/XC non-denaturing loading buffer
40-3004-01
100 µl
5.00
5X Orange G/XC non-denaturing loading buffer
40-3004-10
1ml
10.00
2X BPB/XC Sequencing loading buffer
40-5027-01
100 µl
5.00
2X BPB/XC Sequencing loading buffer
40-5027-10
1ml
10.00
113
Omni-Pure™ DNA & RNA Purification Systems
Facile and rapid purification of DNA and RNA from varied sources
can be performed using the Omni-Pure™ series of DNA, RNA and
plasmid purification system.
♦Omni-Pure™ Genomic DNA Purification System
Each purification sample volume is specially geared towards the
desired downstream application. A sample volume of 300 µl is
recommended for human blood samples yielding on average from
5-15 µg of high molecular weight and high quality genomic DNA
for 2 restriction digestions for Southern blot analysis. The yield is
sufficient for hundreds of PCR amplification reactions. Product
manual contains detailed protocol for extraction of genomic DNA
from tissues and other bodily fluids.
Genomic DNA purified using 300µl human blood.
♦Omni-Pure™ Viral DNA & RNA Purification Systems
Pathogen infection by either DNA or RNA viruses can be easily
detected using molecular diagnostic methods using the DNA or
RNA extracted by these systems. Rapid purification systems for
extraction of viral DNA or RNA from human bodily fluids including
blood for detection of Viral DNA or RNA are captured on special
membranes and then eluted in a low volume for direct use in
qualitative and quantitative amplification protocols for detection of
pathogen.
Viral DNA purification and amplification using
zero, 1, 10, 100ng.
♦ Omni-Pure™ Microbial DNA Purification System
The microbial DNA purification system is ideal for DNA purification
of pathogen DNA. An example is mycobacterium tuberculosis;
purification of MTB genomic DNA for sputum and other bodily
fluids are rapidly performed in less than 30 minutes using this
system. The pathogen DNA can be directly used for qualitative
and quantitative amplification protocols for detection of pathogen.
♦ Omni-Pure™ Plasmid DNA Purification System
Microbial DNA purification followed by
amplification of a specific fragment.
Mini-preps of plasmid extraction are achieved in less than 30
minutes using these kits. The purified DNA is of high quality
suitable for all molecular biology applications including direct use
in fluorescent automated sequencing methods. Purification can be
performed with a maximum of 3 ml of cells yielding up to 20 µg of
purified DNA. Convenient spin column method for ease of use and
scale up by using multiple columns.
Plasmid purification using 3 ml cultures
114
Omni-Pure™ DNA & RNA Purification Systems
Product
Catalog No.
Size*
(Purifications)
Price $
Omni-Pure™ Genomic DNA Purification System
40-4010-01
100
75.00
Omni-Pure™ Genomic DNA Purification System
40-4010-05
500
210.00
Omni-Pure™ Genomic DNA Purification System
40-4010-10
1000
350.00
Omni-Pure™ Viral DNA Purification System
40-3720-01
100
220.00
Omni-Pure™ Viral DNA Purification System
40-3720-05
500
880.00
Omni-Pure™ Viral DNA Purification System
40-3720-50
1000
1400.00
Omni-Pure™ Microbial DNA Purification System
40-3700-01
100
175.00
Omni-Pure™ Microbial DNA Purification System
40-3700-05
500
700.00
Omni-Pure™ Microbial DNA Purification System
40-3700-10
1000
1120.00
Omni-Pure™ Viral RNA Purification System
40-3650-01
100
175.00
Omni-Pure™ Viral RNA Purification System
40-3650-05
500
700.00
Omni-Pure™ Viral RNA Purification System
40-3650-10
1000
1120.00
Omni-Pure™ Plasmid DNA Purification System
40-4020-01
100
95.00
Omni-Pure™ Plasmid DNA Purification System
40-4020-05
500
375.00
Omni-Pure™ Plasmid DNA Purification System
40-4020-10
1000
595.00
*Sample volume for each purification system varies. Each purification yield sufficient quantity for desired applications.
Related Products
Omni-Clean™ Gel DNA Purification and Concentration Systems
Product
Catalog No.
Size*
(Purifications)
Price $
Omni-Clean™ Gel DNA Beads Purification System
40-4110-10
100
95.00
Omni-Clean™ Gel DNA Beads Purification System
40-4110-50
500
380.00
Omni-Clean™ Gel DNA Spin Column Purification System
40-4120-10
100
110.00
Omni-Clean™ Gel DNA Spin Column Purification System
40-4120-50
500
440.00
Omni-Clean™ DNA Concentration System
40-4130-10
100
110.00
40-4130-50
500
440.00
Omni-Clean™ DNA Concentration System
*Unit of size is purification performed
Omni-Marker™ unlabeled DNA molecular weight markers*
Product
Catalog No.
Size*
(Purifications)
Price $
Omni-Marker™ Universal unlabeled
40-3005-01
100 µl
15.00
Omni- Marker™ Universal unlabeled
40-3005-05
500 µl
50.00
Omni-Marker™ Universal unlabeled
40-3005-10
1 ml
90.00
Omni- Marker™ Low unlabeled
40-3006-01
100 µl
15.00
Omni-Marker™ Low unlabeled
40-3006-05
500 µl
50.00
Omni- Marker™ Low unlabeled
40-3006-10
1 ml
90.00
* The markers are provided ready to load containing BPB/XC dyes. A loading of 5µl/well is suggested.
Prices subject to change without notice.
All Gene Link products are for research use only.
115
Omni-Clean™ Gel DNA Purification & Concentration Systems
Facile and rapid purification and concentration of DNA excised from gels and for DNA concentration for
sequencing and genotyping. The Omni-Clean™ System complement’s the Omni-Pure™ DNA purification
system product line.
♦ Omni-Clean™
System
Gel
DNA
Beads
Purification
This system utilizes specialized beads which minimizes
shearing of large molecular weight genomic DNA. This
system is thus ideal for large fragments. The binding
capacity is almost 20µg per purification. Purified DNA
is suitable for all molecular biology applications.
Lane 1 is plasmid extracted using Omni-Pure™plasmid
purification system. Lane 2 is the lower fragment gel purified
using the Omni-Clean™ gel bead based purification system.
♦ Omni-Clean™ Gel DNA Column Purification
System
The spin column based gel DNA purification system
yields ultra clean DNA without the possibility of carry
over of beads during the pipeting process. The binding
capacity is almost 20µg per column. Purified DNA is
suitable for all molecular biology applications.
Lane 2 and 3 are fragments excised from agarose gel and
purified using the Omni-Clean™ column based purification
system.
♦ Omni-Clean™ DNA Concentration System
Concentration of dilute DNA samples by ethanol
precipitation is tedious and there is inevitable salt
carry over. The Omni-Clean™ DNA concentration
system takes less than 15 minutes and yields ultra
high quality DNA suitable for all molecular biology
applications.
This system is recommended for regular ultra
purification in addition to concentration. DNA
concentrated using this system yields fail safe data for
automated sequencing and genotyping.
Electropherograms of typical DNA purified using the OmniClean™ DNA concentration system
116
Omni-Clean™ Gel DNA Purification and Concentration Systems
Product
Catalog No.
Size*
(Purifications)
Price $
Omni-Clean™ Gel DNA Beads Purification System
40-4110-10
100
95.00
Omni-Clean™ Gel DNA Beads Purification System
40-4110-50
500
380.00
Omni-Clean™ Gel DNA Spin Column Purification System
40-4120-10
100
110.00
Omni-Clean™ Gel DNA Spin Column Purification System
40-4120-50
500
440.00
Omni-Clean™ DNA Concentration System
40-4130-10
100
110.00
Omni-Clean™ DNA Concentration System
40-4130-50
500
440.00
*Unit of size is purification performed
Related Products
Omni-Pure™ DNA & RNA Purification Systems
Product
Catalog No.
Size*
(Purifications)
Price $
Omni-Pure™ Genomic DNA Purification System
40-4010-01
100
75.00
Omni-Pure™ Genomic DNA Purification System
40-4010-05
500
210.00
Omni-Pure™ Genomic DNA Purification System
40-4010-10
1000
350.00
Omni-Pure™ Viral DNA Purification System
40-3720-01
100
220.00
Omni-Pure™ Viral DNA Purification System
40-3720-05
500
880.00
Omni-Pure™ Viral DNA Purification System
40-3720-50
1000
1400.00
Omni-Pure™ Microbial DNA Purification System
40-3700-01
100
175.00
Omni-Pure™ Microbial DNA Purification System
40-3700-05
500
700.00
Omni-Pure™ Microbial DNA Purification System
40-3700-10
1000
1120.00
Omni-Pure™ Viral RNA Purification System
40-3650-01
100
175.00
Omni-Pure™ Viral RNA Purification System
40-3650-05
500
700.00
Omni-Pure™ Viral RNA Purification System
40-3650-10
1000
1120.00
Omni-Pure™ Plasmid DNA Purification System
40-4020-01
100
95.00
Omni-Pure™ Plasmid DNA Purification System
40-4020-05
500
375.00
Omni-Pure™ Plasmid DNA Purification System
40-4020-10
1000
595.00
*Sample volume for each purification system varies. Each purification yield sufficient quantity for desired applications.
Omni-Marker™ unlabeled DNA molecular weight markers*
Product
Catalog No.
Size*
(Purifications)
Price $
Omni-Marker™ Universal unlabeled
40-3005-01
100 µl
15.00
Omni- Marker™ Universal unlabeled
40-3005-05
500 µl
50.00
Omni-Marker™ Universal unlabeled
40-3005-10
1 ml
90.00
Omni- Marker™ Low unlabeled
40-3006-01
100 µl
15.00
Omni-Marker™ Low unlabeled
40-3006-05
500 µl
50.00
Omni- Marker™ Low unlabeled
40-3006-10
1 ml
90.00
* The markers are provided ready to load containing BPB/XC dyes. A loading of 5µl/well is suggested.
Prices subject to change without notice.
All Gene Link products are for research use only.
117
Guinea Pig First Strand cDNA
Background
First strand cDNA is useful for amplifying a particular cDNA using PCR. The PCR reaction must be optimized
using varying amounts of the cDNA. This optimization is particularly important when the target mRNA species is
of low abundance. The protocol given is for amplifying β-actin as a control to validate the quality of the ‘first
strand cDNA’ supplied. The PCR conditions to amplify the target cDNA will be based on the primers selected. It
should be noted that specific sequence primers as well as degenerate sequence primers can be used
successfully to amplify the target sequence.
The first strand cDNA has been prepared from freshly obtained tissue and appropriately frozen during
transportation. RNA was extracted using the widely used and published method (1). Oligo dT has been used to
prime the synthesis of the first strand using Moloney Murine leukemia Virus (MMLV) Reverse Transcriptase. The
amount supplied, 5 µg (lyophilized) first strand cDNA and 200µl of β-actin control PCR mix, is sufficient for at
least 50 amplifications. Each lot is tested for amplification of β-actin cDNA.
An amplified fragment of 289 bp. Lane 1 is molecular weight markers. Lanes 2-6 are β-actin control PCR
product from brain, liver, intestine, skeletal muscle and spleen.
References
1. Chomczynski,P. and Sacchi, N. (1987) Anal. Biochem. 162:156-159.
First Strand cDNA
Catalog No.
Size
Price $
Guinea pig first strand pooled cDNA
10-2100-05
5µg
425.00
Guinea pig first strand cDNA, Brain
10-2101-05
5µg
425.00
Guinea pig first strand cDNA, Heart
10-2102-05
5µg
425.00
Product
Guinea pig first strand cDNA, Liver
10-2103-05
5µg
425.00
Guinea pig first strand cDNA, Kidney
10-2104-05
5µg
425.00
Guinea pig first strand cDNA, Intestine
10-2105-05
5µg
425.00
Guinea pig first strand cDNA, Skeletal muscle
10-2106-05
5µg
425.00
Guinea pig first strand cDNA, Lungs
10-2107-05
5µg
425.00
Guinea pig first strand cDNA, Spleen
10-2108-05
5µg
425.00
Guinea pig first strand cDNA, Ovaries
10-2109-05
5µg
425.00
Guinea pig first strand cDNA, Pancreas
10-2110-05
5µg
425.00
Guinea pig first strand cDNA, Eye
10-2111-05
5µg
425.00
118
Omni-cDNA™
Pooled First Strand cDNA
First strand cDNA is useful for amplifying a particular cDNA using PCR. The PCR reaction must be optimized
using varying amounts of the cDNA. This optimization is particularly important when the target mRNA species is
of low abundance. The protocol given is for amplifying β-actin as a control to validate the quality of the ‘first
strand cDNA’ supplied. The PCR conditions to amplify the target cDNA will be based on the primers selected. It
should be noted that specific sequence primers as well as degenerate sequence primers can be used
successfully to amplify the target sequence.
The first strand cDNA has been prepared from pooled and or amplified mRNA obtained from different tissues.
These are not from cultured cell lines. The various tissues vary, but are representative of different organs and
tissue types. These include lung, heart, brain, spleen, skeletal muscle, smooth muscle, ovaries, pancreas, liver
and kidney. There is lot to lot variation but an overall representation of tissue type is maintained. Oligo dT has
been used to prime the synthesis of the first strand using Moloney Murine leukemia Virus (MMLV) Reverse
Transcriptase or AMV reverse transcriptase. The amount supplied is sufficient for at least 50 amplifications.
Each lot is tested for amplification of β-actin cDNA.
Omni-cDNA™ pooled first strand size distribution is from ~5kb to 200bp. These can also be used for cloning
mRNA of interest by RT-PCR. A 1.3 kb and a ~500bp amplified cDNA fragment of p53 is shown in the figure.
β-actin amplified fragment of 289 bp. Lane 1 is
molecular weight markers. Lanes 2-5 are β-actin
control PCR product from guinea pig, human,
mouse and rat pooled first strand Omni-cDNA™.
p53 cDNA amplification from human Omni-mRNA™
pooled reference mRNA. Lane 1, molecular weight
markers; lanes 2 and 4, ~1.3kb 5’ end fragment of
p53; lane 3 and 5, ~500 bp of middle portion of
p53. Lanes 2-3 and 4-5 represent reproducible
different preparations.
First Strand pooled cDNA
Product
Catalog No.
Size
Price $
Omni-cDNA™
Human first strand pooled cDNA
10-0100-05
5µg
425.00
Omni-cDNA™
Mouse first strand pooled cDNA
10-0200-05
5µg
425.00
Omni-cDNA™
Rat first strand pooled cDNA
10-0300-05
5µg
425.00
Omni-cDNA™
Guinea Pig first strand pooled cDNA
10-2100-05
5µg
425.00
119
Omni-mRNA™ pooled reference mRNA
Gene
Link introduces
Omni-mRNA™
pooled
reference mRNA. Commercially available for the
first time, Omni-mRNA™ is a unique blend of
amplified high-quality mRNA purified from various
tissues. Using the same reference mRNA in different
microarray experiments provides a common
denominator
for
accurate
and
reproducible
comparison of gene expression data. In addition,
use of the same reference mRNA among different
research
groups
allows
inter-laboratory
comparisons as well. Gene Link recommends using
pooled reference mRNA as a reference sample in
any multicolor hybridization experiment using cDNA
or oligonucleotide microarrays.
p53 cDNA amplification from human OmnimRNA™ pooled reference mRNA. Lane 1,
molecular weight markers; lanes 2 and 4,
~1.3kb 5’ end fragment of p53; lane 3 and
5, ~500 bp of middle portion of p53. Lanes
2-3 and 4-5 represent reproducible different
preparations.
Omni-mRNA™
pooled
reference
mRNA
are
compatible with all commercially available labeling
systems. Other applications of pooled reference
mRNA include RNA ELISA, Quantigene, HPSA, and a
number of other RNA amplification/detection
systems.
Omni-mRNA™
pooled
reference
mRNA
size
distribution is from ~5kb to 200bp. These can also
be used for cloning mRNA of interest by RT-PCR. A
1.3 kb and a ~500bp amplified cDNA fragment of
p53 is shown in the figure.
Guinea Pig β-actin amplification. An amplified
fragment of 289 bp. Lane 1 is molecular weight
markers. Lanes 2-6 are β-actin control PCR product
from brain, liver, intestine, skeletal muscle and spleen
first strand cDNA.
Omni-mRNA™ amplified pooled reference mRNA
Quantity supplied 50 µg in 25 µg x 2 tubes is sufficient for direct hybridization of 20 microarrays
Product
Human Omni-mRNA™ amplified pooled reference mRNA
Catalog No.
Size
Price $
08-0100-50
50µg (25µg x 2 tubes)
395.00
Mouse Omni-mRNA™ amplified pooled reference mRNA
08-0200-50
50µg (25µg x 2 tubes)
395.00
Rat Omni-mRNA™ amplified pooled reference mRNA
08-0300-50
50µg (25µg x 2 tubes)
395.00
Guinea Pig Omni-mRNA™ amplified pooled reference mRNA
08-2100-50
50µg (25µg x 2 tubes)
395.00
120
Omni-Array™ Amplification Kits
When the availability of total RNA becomes the limiting factor in performing certain experimental procedures,
the Omni-RNA Amplification Kit provides a rapid and simple procedure for the generation of usable amounts of
high quality sense or antisense strand RNA. The amplified RNA is suitable for microarrays, RT-PCR, cloning, in
vitro transcription, and a multitude of other applications. Using this amplification protocol, microgram quantities
of sense or antisense RNA can be produced from as little as 2 ng of total RNA in a single round of amplification.
The Omni-Array system offers the user two protocols for amplification of sense or antisense strand RNA
depending on the initial amount of total RNA present. A single round protocol is sufficient to generate > 10µg of
sense or antisense strand RNA from 100 ng of total RNA. When the initial amount of total RNA is less than 100
ng, a two round amplification protocol is recommended. Using two rounds of amplification, > 10 µg of sense or
Antisense strand RNA can be generated from as little as 2 ng of total RNA. The single round protocol can easily
be performed in less than 1 day while the 2 round protocol requires approximately 1 ½ days.
Omni-Array™ Amplification Strategy
Omni-Array™ mRNA Amplification Kits
Catalog No.
Size
Price $
Omni-Array ™ Sense strand mRNA amplification kit, 2 ng
Version
Product
08-0011-02
10 rxns
495.00
Omni-Array ™ Antisense strand mRNA amplification kit, 2ng
Version
08-0021-02
10 rxns
495.00
121
122
Appendix
123
124
Amino Acid Abbreviations
Amino acid
3 letter abrv.
1 letter abrv.
MW
Alanine
Ala
A
89
Arginine
Arg
R
174
Asparagine
Asn
N
132
Aspartic Acid
Asp
D
133
Cysteine
Cys
C
121
Glutamic Acid
Glu
E
147
Glutamine
GlN
Q
146
Glycine
Gly
G
75
Histidine
His
H
155
Isoleucine
Ile
I
131
Leucine
Leu
L
131
Lysine
Lys
K
146
Methionine
Met
M
149
Phenylalanine
Phe
F
165
Proline
Pro
P
115
Serine
Ser
S
105
Threonine
Thr
T
119
Tryptophan
Trp
W
204
Tyrosine
Tyr
Y
181
Valine
Val
V
117
125
The Standard DNA Genetic Code
First
Position
(5' end)
T
C
A
T
C
A
G
TTT Phe [F]
TCT Ser [S]
TAT Tyr [Y]
TGT Cys [C]
T
TTC Phe [F]
TCC Ser [S]
TAC Tyr [Y]
TGC Cys [C]
C
TTA Leu [L]
TCA Ser [S] TAA Stop [end] TGA Stop [end]
A
TTG Leu [L]
TCG Ser [S] TAG Stop [end]
G
TGG Trp [W]
CTT Leu [L]
CCT Pro [P]
CAT His [H]
CGT Arg [R]
T
CTC Leu [L]
CCC Pro [P]
CAC His [H]
CGC Arg [R]
C
CTA Leu [L]
CCA Pro [P]
CAA Gln [Q]
CGA Arg [R]
A
CTG Leu [L]
CCG Pro[P]
CAG Gln [Q]
CGG Arg [R]
G
ATT Ile [I]
ACT Thr [T]
AAT Asn [N]
AGT Ser [S]
T
ATC Ile [I]
ACC Thr [T]
AAC Asn [N]
AGC Ser [S]
C
ATA Ile [I]
ACA Thr [T]
AAA Lys [K]
AGA Arg [R]
A
ACG Thr [T]
AAG Lys [K]
AGG Arg [R]
G
ATG Met [M]
Start
G
Third Position
(3' end)
Second Position
GTT Val [V]
GCT Ala [A]
GAT Asp [D]
GGT Gly [G]
T
GTC Val [V]
GCC Ala [A]
GAC Asp [D]
GGC Gly [G]
C
GTA Val [V]
GCA Ala [A]
GAA Glu [E]
GGA Gly [G]
A
GTG Val [V]
GCG Ala [A]
GAG Glu [E]
GGG Gly [G]
G
Start Codon
Stop Codon
Nonpolar Side Chain
Uncharged Polar Side Chain
Charged Polar Side Chain
IUB Standard Amino Acid Codes
[A] Ala: Alanine
[C] Cys: Cysteine
[D] Asp: Aspartic acid
[E] Glu: Glutamic acid
[F] Phe: Phenylalanine
[G ] Gly: Glycine
[H] His: Histidine
[I] Ile: Isoleucine
[K] Lys: Lysine
[L] Leu: Leucine
[M] Met: Methionine
[N] Asn: Asparagine
[P] Pro: Proline
[Q] Gln: Glutamine
[R] Arg: Arginine
[S] Ser: Serine
[T] Thr: Threonine
[V] Val: Valine
[W] Trp: Tryptophan
[Y] Tyr: Tyrosine
126
Common Conversions of Nucleic Acids
Molar Conversions
1µg of 1000 bp DNA = 1.52pmol
1µg of pUC18/19 DNA (2686 bp) = 0.57pmol
1µg of pBR322 DNA (4361 bp) = 0.35pmol
1µg of SV40 DNA (5243 bp) = 0.29pmol
1µg of PhiX174 DNA (5386 bp) = 0.28pmol
1µg of M13mp18/19 DNA (7250 bp) = 0.21pmol
1µg of lambda phage DNA (48502 bp) = 0.03pmol
1pmol of 1000 bp DNA = 0.66µg
1pmol of pUC18/19 DNA (2686 bp) = 1.77µg
1pmol of pBR322 DNA (4361 bp) = 2.88µg
1pmol of SV40 DNA (5243 bp) = 3.46µg
1pmol of PhiX174 DNA (5386 bp) = 3.54µg
1pmol of M13mp18/19 DNA (7250 bp) = 4.78µg
1pmol of lambda phage DNA (48502 bp) = 32.01µg
Spectrophotometric Conversions
1 A260 of dsDNA = 50µg/ml = 0.15mM (in nucleotides)
1 A260 of ssDNA = 33µg/ml = 0.1mM (in nucleotides)
1 A260 of ssRNA = 40µg/ml = 0.12mM (in nucleotides)
1mM (in nucleotides) of dsDNA = 6.7 A260 units
1mM (in nucleotides) of ssDNA = 10.0 A260 units
1mM (in nucleotides) of ssRNA = 8.3 A260 units
The average MW of a deoxyribonucleotide base = 333 Daltons
The average MW of a ribonucleotide base = 340 Daltons
Reference
1.
Sambrook, J. et al,. (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory. Cold Spring
Harbor, N.Y.
127
Estimation of Ends (3’ or 5’) Concentration
Circular DNA
pmol ends = pmol DNA x number of cuts x 2
Linear DNA
pmol ends = pmol DNA x (number of cuts x 2 + 2)
1µg of 1000 bp DNA = 3.04pmol ends
1µg of linear pUC18/19 DNA = 1.14pmol ends
1µg of linear pBR322 DNA = 0.7pmol ends
1µg of linear SV40 DNA = 0.58pmol ends
1µg of linear PhiX174 DNA = 0.56pmol ends
1µg of linear M13mp18/19 DNA = 0.42pmol ends
1µg of lambda phage DNA = 0.06pmol end
Common Conversions of Oligonucleotides
Molecular Weight
MW = 333 x N
Concentration of Oligonucleotides
C (µM or pmol/µl) = A260 / (0.01 x N) C (ng/ml) = (A260 x MW) / (0.01 x N)
MW - molecular weight, Da
A260 - absorbance at 260nm
N - number of bases
Melting Temperature of Duplex DNA and Oligonucleotides
For Duplex Oligonucleotide shorter than 25 bp (1)
Tm = 2(A+T) + 4(C+G)
A, T, C, G - number of respective bases.
Presence of m5C in oligonucleotide increases the melting temperature of
duplex. m4C and m6A have an opposite effect (2, 3).
For Duplex DNA longer than 25 bp (4)
Tm=81.5°C+16.6log(MNaCl)+0.41(%GC)-(500/N)-0.65(%formamide)
N - number of bp
MNaCl - molar concentration of NaCl
References
1. Thein, S.L., Wallance, R.B., Human Genetic Diseases: a practical approach, IRL Press, Herndon, Virginia, 33-50,
1986.
2. Butkus, V., Klimasauskas, S., Petrauskiene, L., Maneliene, Z., Janulaitis, A., Minchenkova, L.E. and Schyolkina, A.K.,
Nucleic Acids Res., 20, 8467-8478, 1987.
3. Jurgaitis, A., Butkus, V., Klimasauskas, S., Janulaitis, A., Bioorganicheskaya Khimiya, 14, 158-165, 1988.
4. Bolton, E.T., McCarthy, B.J., Proc. Natl. Acad. Sci. USA 48, 1390-1397, 1962.
128
Commonly Used Media, Stock Solutions and Buffers
Growth Media
Stock Solutions
LB Medium, per liter:
Tryptone
Yeast extract
NaCl
H2 O
Adjust pH to 7.0
10g Ammonium acetate
5g H2O
10g
to 1 liter
10g CaCl2x2H2O
5g H2O
5g
to 1 liter
12g
24g
4ml
to 900ml
Ficoll 400
Polyvinylpyrrolidone
Bovine serum albumin
H2 O
Filter sterilize and store at 20°C in 25ml aliquots.
SOB Medium (1 liter) with the addition of 20ml
filter sterilized 1M glucose.
Na2EDTAx2H2O
H2 O
Adjust pH to 8.0 with 10M
NaOH
H2 O
M9 Minimal Medium, per liter:
10mg/ml Ethidium Bromide:
5X M9 Salts, per liter:
200ml
to 1 liter
2ml
20ml
0.1ml
Ethidium bromide
H2 O
Mix well and store at 4°C in
dark.
CAUTION: Ethidium
bromide is a mutagen and
must be handled carefully.
1M KCl:
175.3g (3M)
88.2g (0.3M)
to 800ml
20X SSPE, per liter:
10g
10g
10g
to 500ml
NaCl
NaH2PO4xH2O
Na2EDTA
H2 O
Adjust pH to 7.4 with 10M
NaOH
H2O to 1 liter
175.3g (3M)
27.6g (0.2M)
7.4g (0.02M)
to 800ml
5X SDS Electrophoresis Buffer, per liter:
15.45g Tris base
to 100ml Glycine
SDS
H2 O
Dilute to 1X or 2X for
working solution, as
appropriate.
Store up to 1 month at 0°C
to 4°C.
Do not adjust the pH of the
solution, as the solution is
pH 8.3 when diluted.
0.5M EDTA (ethylenediamine
tetraacetic acid) (pH 8.0):
SOC Medium, per liter:
5X M9 salts
Sterile H2O
1M MgSO4
20% glucose
1M CaCl2
147g NaCl
to 1 liter Na3citratexH2O
H2 O
Adjust pH to 7.0 with 1M HCl
H2O to 1 liter
1M Dithiothreitol (DTT):
20g DTT
5g H2O
0.5g Store at -20°C
10ml
to 900ml
80g
2g
14.4g
2.4g
to 800ml
to pH 7.4
to 1 liter
20X SSC, per liter:
100X Denhardt Solution:
SOB Medium, per liter:
Tryptone
Yeast extract
NaCl
250mM KCl
H2 O
Adjust pH to 7.0 and add H2O
to 990ml.
Autoclave, cool to room
temperature and add 10ml of
sterile solution of 1M MgCl2
before use.
385.4g NaCl
to 500ml KCl
Na2HPO4
KH2PO4
H2 O
HCl
H2 O
1M CaCl2:
Terrific Broth Medium, per liter:
Tryptone
Yeast extract
Glycerol
Add H2O
Autoclave, cool to 60°C or less
before adding 100ml of filter
sterilized 10X TB phosphate
(0.17M KH2PO4, 0.72M
K2HPO4).
10X Stock Phosphate-buffered Saline (PBS),
per liter:
10M Ammonium Acetate:
Low Salt LB Medium, per liter:
Tryptone
Yeast extract
NaCl
H2 O
Adjust pH to 7.0
Buffers
15.1g
72.0g
5.0g
to 1 liter
50X TAE (Tris/acetate/EDTA)
Electrophoresis Buffer, per liter:
186.1g
to 700ml
(~50ml)
to 1 liter
Tris base
Glacial acetic acid
0.5M EDTA (pH 8.0)
H2 O
Adjust pH to ~8.5
242g
57.1ml
100ml
to 1 liter
10X TBE (Tris/borate/EDTA) Electrophoresis
Buffer, per liter:
0.2g Tris base
to 20ml Boric acid
0.5M EDTA (pH 8.0)
H2 O
108g
55g
40ml
to 1 liter
10X TPE (Tris/phosphate/EDTA)
129
Electrophoresis Buffer, per liter:
Na2HPO4x7H2O
KH2PO4
NaCl
NH4Cl
64g KCl
15g H2O
2.5g
5g
Additives
to
to
to
to
MgCl2x6H2O
50µg/ml H2O
20µg/ml
30µg/ml
12µg/ml
Galactosides:
X-Gal
IPTG
to 20µg/ml
to 0.1mM
Media containing agar or agarose:
108g
15.5ml
40ml
to 1 liter
TE (Tris/EDTA) Buffer, pH 7.4, 7.6 or 8.0,
per liter:
1M MgCl2:
Antibiotics:
Ampicillin
Chloramphenicol
Kanamycin
Tetracycline
Agar (for plates)
Agar (for top agar)
Agarose (for plates)
Agarose (for top agarose)
74.6g Tris base
to 1 liter Phosphoric acid (85%)
0.5M EDTA (pH 8.0)
40ml
20.3g 1M Tris, pH 7.4, 7.6 or 8.0
to 100ml 0.5M EDTA (pH 8.0)
H2 O
10ml (10mM)
2ml (1mM)
to 1 liter
1M MgSO4:
15g
7g
15g
7g
per
per
per
per
liter MgSO4x7H2O
liter H2O
liter
liter
24.6g
to 100ml
5M NaCl:
NaCl
H2 O
292g
to 1 liter
10M NaOH:
NaOH
H2 O
400g
to 1 liter
1M Tris-HCl
[tris(hydroxymethyl)aminomethane]:
Tris base
H2 O
Adjust to desired pH with
concentrated HCl. Mix and
add H2O to 1 liter
121g
to 800ml
3M Sodium Acetate (pH 5.2 and 7.0)
(1):
Sodium acetate. 3H2O
H2O. Adjust the pH to 5.2
with glacial acetic acid or
adjust the pH to 7.0 with
dilute acetic acid. Mix and
add H2O to 1 liter
408.1g
to 800ml
References
1. Sambrook, J., Fritch, E.F., Maniatis, T.,Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, A.1-B.25, 1989.
2. Current Protocols in Molecular Biology, vol. 1 (Ausubel, F.M., et al., ed.), John Wiley & Sons, Inc., Brooklyn, New
York, 1.1.1-1.1.4, 1999.
3. Current Protocols in Molecular Biology, vol. 4 (Ausubel, F.M., et al., ed.), John Wiley & Sons, Inc., Brooklyn, New
York, A.2.1-A.2.6, 1999.
130
DNA Migration in Agarose and Polyacrylamide Gels
Recommended Gel Percentages for Separation of Linear DNA
Agarose
gel,%
0.5
Gel,%
Range of
separation,bp
1,000-30,000
Polyacrylamide gel,%
3.5
Range of
separation,bp
100-1,000
0.7
800-12,000
5.0
80-500
1.0
500-10,000
8.0
60-400
1.2
400-7,000
12.0
40-200
1.4
200-4,000
20.0
5-100
2.0
50-2,000
Dye Migration in Polyacrylamide Non-denaturing Gels
Bromophenol blue
Xylene cyanol
(Size of the fragmen
in nucleotides)
460
3.5
100
5.0
65
260
8.0
45
160
12.0
20
70
15.0
15
60
20.0
12
45
Gel,%
5.0
Dye Migration in Polyacrylamide Denaturing Gels
Bromophenol blue
Xylene cyanol
(Size of the fragments
in nucleotides)
35
140
6.0
26
106
8.0
19
75
10.0
12
55
20.0
8
28
Reference
1.
Sambrook, J., et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory. Cold Spring Harbor,
N.Y., 1989.
DNA Size Migration with Sample Loading Dyes
Agarose
Xylene cyanol
Bromophenol
Orange G
concentration,%
blue
0.7-1.7
~4000bp
~300bp
~50bp
2.5-3.0
~800bp
~100bp
~30bp
131
International System of Unit Prefixes
Prefix
Symbol
Multiple
exa
(E)
10E18
peta
(P)
10E15
tera
(T)
10E12
giga
(G)
10E9
mega
(M)
10E6
kilo
(k)
10E3
hecto
(h)
10E2
deka
(da)
10E1
deci
(d)
10E-1
centi
(c)
10E-2
milli
(m)
10E-3
micro
(µ)
10E-6
nano
(n)
10E-9
pico
(p)
10E-12
femto
(f)
10E-15
atto
(a)
10E-18
132
Physical Constants of the Nucleoside Triphosphates and Related Compounds
Compound
MW
lambdamax*,nm
ec**
ATP
507
259
15400
CTP
483
271
9000
GTP
523
253
13700
UTP
484
262
10000
dATP
491
259
15200
dCTP
467
271
9300
dGTP
507
253
13700
dTTP
482
267
9600
ddATP
475
261
15200
ddCTP
451
281***
13100
ddGTP
491
253
13600
ddTTP
466
267
9600
NAD
664
260
18000
NADH
665
338****
NADP
743
260
18000
NADPH
745
260
18000
6200
* determined at pH 7.0
** extinction coefficient (absorbance at lambdamax for 1M solution at pH 7.0)
*** determined at pH 2.0
**** determined at pH 10.0
Conversion Formula
C = A / e x 10E3
C - mM concentration of compounds
A - observed absorbance at lambdamax
e - extinction coefficient
133
Physical Properties of Some Common Radioisotopes
Radioisotope
Half-life
Specific Activity (MBq/mmol)
32
14.3 days
10E2-10E7
P
25.4 days
10E2-10E7
S
87.4 days
10-10E7
33
35
P
131
I
8.06 days
10E3-10E5
125
I
60 days
10E3-10E7
C
5730 years
10-10E3
H
12.43 years
10E3-10E6
14
3
Summary of Useful Conversion
1Becquerel (Bq) = 1 disintegration per second = 2.7x10E-11Curies (Ci)
1Ci = 3.7x10E10Bq = 37GBq = 2.22x10E12 disintegrations per minute (dpm)
1mCi = 37MBq = 2.22x10E9dpm
1µCi = 37kBq = 2.22x10E6dpm
1GBq = 27mCi
1MBq = 27µCi
1kBq = 27nCi
134
Size and MW of Various Nucleic Acids
Nucleic acid
Length in bases or base pairs
MW, Daltons
75
2.5 x 10E4
120
3.6 x 10E4
16S rRNA
1700
5.5 x 10E5
18S rRNA
1900
6.1 x 10E5
23S rRNA
3700
1.2 x 10E6
28S rRNA
4800
1.6 x 10E6
pBR322 DNA
4361
2.8 x 10E6
SV40
5243
3.5 x 10E6
PhiX174
5386
3.6 x 10E6
Adenovirus 2 (Ad2)
35937
2.8 x 10E7
Lambda phage
48502
3.1 x 10E7
Escherichia coli
4.7 x 10E6
3.1 x 10E9
Saccharomyces cerevisiae
1.5 x 10E7
9.9 x 10E9
Dictyostelium discoideum
5.4 x 10E7
3.6 x 10E10
Arabidopsis thaliana
7.0 x 10E7
4.6 x 10E10
Caenorhabditis elegans
8.0 x 10E7
5.3 x 10E10
Drosophila melanogaster
1.4 x 10E8
9.2 x 10E10
Gallus domesticus (chicken)
1.2 x 10E9
7.9 x 10E11
Mus musculus (mouse)
2.7 x 10E9
1.8 x 10E12
Rattus norvegicus (rat)
3.0 x 10E9
2.0 x 10E12
Xenopus laevis
3.1 x 10E9
2.0 x 10E12
Homo sapiens
3.3 x 10E9
2.2 x 10E12
Zea mays
3.9 x 10E9
2.6 x 10E12
Nicotiana tabacum
4.8 x 10E9
3.2 x 10E12
RNA
tRNA (E.coli)
5S rRNA
DNA
Reference
1.
Ausubel, F.M., et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York, 1988.
135
Temperature Dependence of the pH for Commonly Used Buffers
Buffer System
pKa/20°C
[Delta]pKa/10°C
MES
6.15
-0.110
ADA
6.60
-0.110
PIPES
6.80
-0.085
ACES
6.90
-0.200
BES
7.15
-0.160
MOPS
7.20
-0.013
TES
7.50
-0.200
HEPES
7.55
-0.140
TRICINE
8.15
-0.210
TRIS
8.30
-0.310
BICINE
8.35
-0.180
GLYCYLGLYCINE
8.40
-0.280
Reference
1.
Good, N.E., Biochemistry 5, 467-476, 1986.
Temperature Dependence of the pH of 50mM Tris-HCl Solutions
4°C
25°C
37°C
8.1
7.5
7.2
8.2
7.6
7.3
8.3
7.7
7.4
8.4
7.8
7.5
8.5
7.9
7.6
8.6
8.0
7.7
8.7
8.1
7.8
8.8
8.2
7.9
8.9
8.3
8.0
9.0
8.4
8.1
9.1
8.5
8.2
9.2
8.6
8.3
9.3
8.7
8.4
9.4
8.8
8.5
136
DNA Extinction Coefficients
Nucleotide
(A260 / mol)
A
15.4
T
8.7
G
11.5
C
7.4
I
7.2**
I
10.7*
Neighbors (A260 / mol)
AA
13.7
AT
11.4
AG
12.5
AC
10.6
AI
9.3**
AN
12.2*
TA
11.7
TT
8.4
TG
9.5
TC
8.1
TI
8.1**
TN
9.4*
GA
12.60
GT
10.0
GG
10.8
GC
8.8
GI
8.8**
GN
10.5*
CA
10.6
CT
7.6
CG
9.0
CC
7.3
CI
7.2**
CN
8.6*
IA
9.3**
IT
8.4**
IG
8.8**
IC
7.1**
II
6.8**
IN
8.4*
NA
12.1*
NT
9.4*
NG
9.4*
NC
8.7*
NI
8.7*
NN
9.9*
Handbook of Biochemistry and Molecular Biology (1975) Fasman G.D., ed., 3rd edition, Nucleic Acids - Vol. 1, pp 589, CRC Press, Cleveland, OH.
137
MW and TM Calculation
Base
BaseAbbreviation
MW
EC
DeoxyAdenosine
A
313.21
15.4
DeoxyCytosine
C
289.19
7.4
DeoxyGuanosine
G
329.21
11.5
Thymidine
T
304.2
8.7
Inosine
I
314.2
7.2
A+G+T+C
N
308.95
10.70
A+G
R
321.21
13.45
C+T
Y
296.69
8.05
A+C
M
301.2
11.40
G+T
K
316.7
10.10
G+C
S
309.2
9.45
A+T
W
308.71
12.05
A+T+C
H
302.2
10.5
G+T+C
B
307.53
9.20
G+A+T
D
315.54
11.86
G+A+C
V
310.53
11.43
phosphate
P
79.98
0
Other
X
0
0
deoxy uridine
U
290.17
9.9
Notes
add mw of the Modification
138
Formulas for Oligonucleotides
Size= Total number of bases.
%GC= (G+C)/Size
mw = (A x 313.2) + (C x 289.19 ) + (G x 329.21 ) + (T x 304.2 ) + (I x 314.2 ) + (N x 308.95
) + (R x 321.21 ) + (Yx 296.69 ) + (M x 301.2) + (K x 316.7 ) + (S x 309.2 ) + (W x 308.71 )
+ (H x 302.2 ) + (B x 307.53 ) + (D x315.54 ) + (V x 310.53 ) + (P x 79.98 ) + (U x 290.17 )
–62
Tm For Oligos shorter than 25 bp = 2(A+T) + 4 (C+G)
For longer oligos: Reference Bolton, Et and McCarthy, B.J. (1962) PNAS 48: 139-1397
Tm=81.5 – 16.6 + (0.41 x %GC)) – 600 / size
EC =
Formula for Tm Calculation
Tm = 81.5 + 16.6 x Log10[Na+] + 0.41 (%GC) – 600/size
[Na+] is set to 100 mM
Example: 5’-ATGCATGCATGCATGCATG3’ 20mer; GC=50%; AT= 50%
Tm = 81.5 + 16.6 x Log10[0.100] + 0.41 x 50 – 600/20
Tm = 81.5 - 16.6 + 0.41 x 50 – 600/20
Tm = 81.5 - 16.6 + 20.5 – 30
Tm = 64.9 + 20.5 – 30
Tm = 85.40 – 30
Tm = 55.4oC
Tm for same oligo using 2(A+T) + 4 (C+G)
= 2(5+5) + 4(5+5)
= 2(10) + 4(10)
= 20+ 40
= 60oC
Form
Degenerate Bases in Sequence
Follow IUB single letter nomenclature for degenerate/mixed bases. The use of inosine is
recommended to reduce the number of degeneracies. For degenerate (mixed bases) positions
use the following IUB codes
R=A+G Y=C+T M=A+C K=G+T
S=G+C W=A+T H=A+T+C B=G+T+C
D=G+A+T V=G+A+C N=A+C+G+T.
Inosine=I
139
Media for Bacterial Culture
LB Broth
An all purpose media for the growth of bacterial culture
Tryptone (casein Peptone)
10.0 g/L
Yeast Extract
5.0 g/L
Nacl
5.0 g/L
LB Agar Plates
An all purpose media for the growth of bacteria on plates
Tryptone (casein Peptone)
10.0 g/L
Yeast Extract
5.0 g/L
Nacl
5.0 g/L
Agar
15 g/L
Terrific Broth
Highly enriched culture media
Tryptone (casein Peptone)
12.0 g/L
Yeast Extract
24.0 g/L
K2HPO4
9.4 g/L
KH2PO4
2.2 g/L
Sterilize and then add
Glycerol
8 ml/L
SOB
Media for competent cell manipulation
Tryptone (casein Peptone)
20.0 g/L
Yeast Extract
5.0 g/L
Nacl
0.5 g/L
MgSO4
5.0 g/L
SOC
Media for the initial propagation of cells after transformation
Tryptone (casein Peptone)
20.0 g/L
Yeast Extract
5.0 g/L
Nacl
0.5 g/L
MgSO4
5.0 g/L
Glucose
3.6 g/L
140
Antibiotics
Ampicillin
Inhibits cell wall synthesis enzymes
Stock Solution
40 mg/ml in H2O
Use at 40µg/ml (ie.1µl of stock/ml medium)
Tetracycline
Binds to 30s ribosomal subunit. Inhibits ribosomal translocation
Stock Solution
10 mg/ml in 50%EtOH
Use at 10-30µg/ml (ie.1-3 µl of stock/ml medium)
Note: Tetracycline HCl can be dissolved in water
Chloramphenicol
Binds to 50s ribosomal subunit and inhibits protein synthesis
Stock Solution
20 mg/ml in 50%EtOH
Use at 20-30µg/ml (ie.2-3µl of stock/ml medium)
Kanamycin
Binds to ribosomal components and inhibits protein synthesis
Stock Solution
10 mg/ml in H2O
Use at 10µg/ml (ie.1µl of stock/ml medium)
141
Protocols
142
Genomic DNA Purification
Genomic DNA is usually extracted from blood. A simple procedure is given below that purifies ~10 µg DNA from
300 µl blood using a 30 minute procedure.
Omni-Pure™ Genomic DNA Purification System
Catalog Number: 40-4010-01
Rapid DNA Purification Protocol for 300 µl Whole Blood
A. Initial Preparation
1. Label two sets of 1.5 ml tubes per sample.
2. Add 900 µl GD-1 solution (RBC Lysis Solution) to one tube for each sample.
3. Add 300 µl Isopropanol (2-propanol) to one tube for each sample. Cap the tubes.
B. Cell Lysis
1. To the tube containing 900 µl GD-1 solution (RBC Lysis Solution) using a filter tip pipet transfer 300 µl whole
blood. Cap and gently mix by inversion. Incubate for 1-3 minutes at room temperature. Mix by inversion a few times
during this incubation period. Incubate longer for fresh blood cells as they are intact and not lysed already.
2. Centrifuge at 3 K rpm for 20 seconds to pellet the white blood cells. A reddish white pellet should be clearly
visible. Decant and discard supernatant leaving behind the last few droplets. Do not totally remove the supernatant.
3. Completely resuspend the white blood cell pellet by vigorously vortexing the tube. Ensure that the pellet is
completely resuspended.
4. To the resuspended cells add 300 µl GD-2 solution (Cell Lysis Solution). Mix by gentle vortexing. You will notice
release of DNA by the thickening of the liquid in the sample. Samples may be stored at this stage for processing
later. It has been shown that the samples are stable in Cell Lysis Solution for at least 2 years at room temperature.
C. Protein Precipitation
1. Add 100 µl GD-3 solution (Protein Precipitation Solution) to the sample in cell lysis solution.
2. Vortex vigorously at for 20 seconds. Small particles of brown color will be appear and be visible at this stage.
3. Centrifuge at 5 K rpm for 1 minute to pellet the precipitated proteins. A clearly visible brown pellet containing
proteins should be collected at the bottom of the tube.
D. DNA Precipitation
1. Decant the supernatant containing the DNA to a new appropriately labeled tube (see initial preparation above)
containing 300 µl 100% Isopropanol (2-propanol).
2. Mix the sample by inversion until a visible white floating DNA strand-particle is identified. 30-40 mixing by
inversion is usually sufficient.
3. Centrifuge at 6 K rpm for 1 minute to collect the DNA as a pellet. A white DNA pellet should be clearly visible.
4. Decant supernatant and place tube inverted on a clean Kimwipe™ tissue paper to drain the remaining
supernatant.
5. To remove residual salts, add 300 µl of 70% ethanol. Vortex gently.
6. Centrifuge at 6 K rpm for 1 minute to collect the DNA as a pellet. Gently take out the tubes so that the pellet is
not dislodged. While holding the tube, rotate tube so that you can watch the pellet. Now carefully decant the ethanol,
keeping an eye on the pellet so that it does not flow away.
7. Place tube inverted on a clean Kimwipe™ tissue paper to drain the remaining ethanol.
8. Air dry the DNA pellet. Do not use vacuum.
E. DNA Reconstitution & Use
1. Add 100 µl of GD-4 solution (DNA Reconstitution Solution). Vortex gently. Incubate at 60°C for 5 minutes to
facilitate dissolution or keep overnight at room temperature.
2. Store DNA at 4 °C. For long-term storage, place sample at –20 °C or –80 °C.
3. Average yield of 10 µg is expected from 300 µl blood DNA. The range is between 5 µg to 15 µg.
4. The 100 µl of purified DNA obtained will have an average concentration of ~ 100 ng/µl.
5. For PCR amplification use 1-2 µl.
6. Use 100 µl for restriction digestion followed by Southern blot analysis.
7. It is convenient to perform multiple 300 µl blood DNA purification instead of scaling up the procedure.
143
Gel Electrophoresis of DNA
Gel electrophoresis of PCR products is the standard method for analyzing reaction quality and yield. PCR products can range
up to 10 kb in length, but the majority of amplifications are at 1 kb and below. Agarose electrophoresis is the classical method
to analyze amplification products from 150 bp to greater than 10 kb. Polyacrylamide gel electrophoresis should be used for
resolution of short fragments in the range of 100 bp to 500 bp when discrimination of as small as a 10 bp difference is
required.
PAGE gels for PCR products formulated with the amount of cross-linker chosen to give pore sizes optimal for the size of DNA
fragment desired. Gels are most often stained in ethidium bromide, even though the fluorescence of this stain is quenched by
polyacrylamide, which decreases sensitivity 2-5 fold. This decrease in sensitivity generally does not present a problem,
because most PCR reactions yield product levels in the microgram range, and ethidium will detect as little as 1/10 of this
amount. Polyacrylamide gels can be stained by silver staining for more sensitive detection.
Agarose Gel Electrophoresis of DNA
Agarose gels are typically run at 20 to 150V. The upper voltage limit is the
amount of heat produced. At room temperature about 5 Watts is correct for a
minigel (Volts x Amps = Watts). At low voltages migration is linearly
proportional to voltage, but long DNA molecules migrate relatively faster in
stronger fields. Migration is inversely proportional to the log of the fragment
length; a log function also governs migration rate and gel concentration (0.5 to
2% for most purposes). Furthermore, supercoiled / circular DNA molecules
migrate at different rates from linear molecules; single-stranded DNA and RNA
migrate at similar rates, but usually faster than double-stranded DNA of the
same length. Salt in the samples increases conductivity and, hence, migration
rate.
The buffers used for most neutral agarose gels (the gel itself and the solution in
which it lies) is 1 x TAE or 1 x TBE. Agarose powder is added to the buffer at
room temperature, heated in a microwave and boiled slowly until the powder
has dissolved. Cast the gel on a horizontal surface once the agarose has been
cooled to ca. 60° C (just cool enough to hold) and add 0.1 µg of ethidium
bromide solution for each ml of gel volume. At times, during removal of the
comb, it is possible to tear the bottom of the sample wells gels, which results in
sample leakage upon loading. This can be avoided by removing the comb after
the gel has been placed in the running buffer.
• Use TAE buffer for most
molecular biology agarose gel
electrophoresis.
1 X TAE Buffer
Agarose Gel Electrophoresis
Buffer
40 mM Tris-Acetate pH 7.8
1 mM EDTA
1 X TBE
Agarose and Polyacrylamide
Gel Electrophoresis Buffer
0.089 M Tris
0.089 M Boric Acid
0.002 M EDTA
Spectrophotometric Determination of DNA Concentration & Estimation by Agarose Gel Electrophoresis
Measuring the optical density (OD) or absorbance at 260 nm (A260) in a UV spectrophotometer is a relatively accurate method
for calculating the concentration of DNA in an aqueous solution if a standard curve is meticulously prepared. An A260 of 1,
using a 1 cm path length, corresponds to a DNA concentration of 50 µg/ml for double stranded DNA, 40 µg/ml for RNA and 33
µg/ml for oligonucleotides. However, this method is not suitable for determining concentrations of dilute solutions of DNA, as
the sensitivity of this method is not very high. For reliable readings, the concentration of double stranded DNA must be
greater than 1 µg/ml. A simple, inexpensive method for the estimation of nanogram quantities of DNA is described in the
following section. We recommend the use of agarose gel electrophoresis for routine approximate determination of DNA
concentration.
The amount of DNA in sample may be estimated by running the sample alongside standards containing known amounts of the
same-sized DNA fragment. In the presence of ethidium bromide staining, the amount of sample DNA can be visually
estimated by comparing the band intensity with that of the known standards.
Ethidium
bromide
is
a
carcinogen. Follow Health and
Safety Procedures established by
your institution.
Follow proper Hazardous Material
Disposal procedures established
by your institution.
An unknown amount of a 4 kb DNA fragment (U) was run alongside known
quantities (indicated in nanograms) of the same DNA fragment. As estimated by
visual comparison with the known standards, the unknown sample contained
240-320 ng of DNA.
•Use 0.1 µg of ethidium bromide
solution for each ml of gel
volume.
144
Polymerase Chain Reaction
PCR Components and Analysis
PCR buffer conditions vary and it is imperative to optimize
buffer conditions for each amplification reaction. At Gene
Link most amplification reactions have been optimized to
work with the following standard buffer condition, unless
otherwise indicated.
dNTP Concentration
Standard dNTP concentration of 0.2 mM of each base is
used. See section on PCR additives when dNTP
concentration is changed.
MgCl2 Concentration
The concentration of Mg++ will vary from 1-5 mM,
depending upon primers and substrate. Since Mg2+ ions
form complexes with dNTPs, primers and DNA templates,
the optimal concentration of MgCl2 has to be selected for
each experiment. Low Mg2+ ions result in a low yield of PCR
product, and high concentrations increase the yield of nonspecific products and promote mis-incorporation. Lower
Mg2+ concentrations are desirable when fidelity of DNA
synthesis is critical. The recommended range of MgCl2
concentration is 1-4 mM, under the standard reaction
conditions specified. At Gene Link, using the standard PCR
buffer with KCl, a final dNTP concentration of 0.2 mM, a
MgCl2 concentration of 1.5 is used in most cases. If the
DNA samples contain EDTA or other chelators, the MgCl2
concentration in the reaction mixture should be raised
proportionally. Given below is an MgCl2 concentration
calculation and addition table using a stock solution of 25
mM MgCl2.
Standard Gene Link PCR
Buffer Composition
10 X PCR buffer
1 X PCR buffer
100 mM Tris-HCl pH 8.3
500 mM KCl
15 mM MgCl2
0.01% Gelatin
10 mM
50 mM
1.5 mM
0.001%
2.0 mM dNTP Stock Solution Preparation*
Component
Volume
100 mM dGTP
100 µl
100 mM dATP
100 µl
100 mM dTTP
100 µl
100 mM dCTP
100 µl
Water
4.6 ml
Total Volume
5 ml
*Aliquot and freeze
MgCl2 Concentration & Addition Table
Final concentration of MgCl2 in 50 µl reaction mix, (mM) 1.0 1.25 1.5 1.75 2.0 2.5 3.0 4.0
Volume of 25 mM MgCl2, µl
Primer Concentration
The final concentration of primers in a PCR reaction is usually 0.5 to 1 µM
(micromolar). This is equivalent to 0.5 to 1 pmol/µl. For a 100 µl reaction
you would add 50 to 100 pmols. At Gene Link we use 0.5 pmol/µl in the
final PCR.
Genemer™ Reconstitution
Stock Primer Mix: Dissolve the supplied 10 nmols of lyophilized
Genemer™ in 100 µl sterile TE. The 10 nmols of primers when dissolved in
100 µl will give a solution of 100 µM i.e. 100 pmols/µl.
2
2.5
3
3.5
4
5
6
8
Always use filter barrier pipette tips
to prevent cross contamination
TE Buffer pH 7.5 Composition
1 X TE Buffer pH 7.5
10 mM Tris-HCl pH 7.5
1 mM EDTA
Primer Mix: Prepare a 10 pmols/µl Primer Mix solution by a ten fold
dilution of the stock primer mix.
Example: Add 180 µl sterile TE to a new tube, to this tube add 20 µl of
primer stock solution. Label this tube as Primer Mix 10 pmols/µl.
Amplification Thermal Cycling
Hot Start: It is essential to have a ‘Hot Start’ profile for amplification of
any fragment from a complex template like human genomic DNA. Taq
polymerase has low activity at room temperature and it is essential to
minimize any mis-priming in the first cycle of amplification. A typical hot
start profile is given below. Various enzyme preparations are available
which are activated by heat in the first cycle. A simple hot start protocol
is given below that can be used with regular Taq polymerase. See the
• Program your thermal cycler
instrument with an amplification profile
prior to beginning the amplification
protocol.
Consult
your
appropriate
instrument manufacturer’s manual.
Typical PCR Premix (/50µl)
145
section on PCR additives for amplification of products from high GC
content templates.
Hot Start
Time &
Temperature
Step
Cycles
Initial
95 oC for 5 minutes
1
Denaturation
Annealing
60 oC Hold Infinity
Hold
Comments: Add Taq premix while on hold.
Amplification File
The initial denaturation step at 94 oC for 30 seconds is sufficient for all
templates. The number of cycles is usually set to 30 and is sufficient to
amplify 1-10 µg of product depending on the initial concentration of
template. A higher number of cycles from 35-45 cycles may be used, but
internal priming on the product and over amplification of unwanted bands
often result from over-cycling. Generally, it is better to focus on
optimizing reaction conditions than to go beyond 35 cycles.
Typical Amplification File
Step
Temperature
Time
Cycles
Denaturation
94 oC
30 sec.
30
Annealing
*
30 sec.
Elongation
72 oC
30 sec.
Fill in Extension
72 oC
7 minutes
1
Hold
4 oC
Infinity
Hold
Based on the Tm of the primers. Usually varies from 50 oC to 65 oC
Component
10 x PCR Buffer
2.0 mM dNTP mix (each)
Primer Mix (10 pmol/µl
each) or 2.5µl of 10
pmol/µl of individual
primer (final 25 pmol of
each primer/50µl)
H2 O
Volume
5 µl
5 µl
2.5 µl
Total Volume
50 µl
37.5 µl
PCR reaction (/50µl)
Component
Volume
PCR premix
45 µl
100ng/µl diluted DNA
1 µl
Hot start and then add
Taq premix
5 µl
Taq Premix EM (/50µl)
Component
Volume
PCR Premix
6 µl
Taq polymerase (5 u/µl)
0.25µl
Add 5 µl/50 µl rxn after initial
denaturation.
Use 2.5 units of Taq for 100 µl reactions.
Taq is usually supplied at a concentration of
5 units/µl
PCR Premix Preparation (PP)
Component
1 X 50 µl Rxn.
Sterile Water
10 X PCR Buffer
2.0 mM dNTP
10 pmol/µl Primer Mix
Taq Enzyme Mix (EM)
32 µl
4.5 µl
5 µl
2.5 µl
5 µl
Template DNA (~500 ng)
1-2 µl
10 X 50 µl
Rxns.
320 µl
45 µl
50 µl
25 µl
50 µl
Add 1-2 µl DNA
to each tube
Total Volume
50 µl
Keep on ice during set up. After adding template start PCR File
• The PCR premix preparation
protocol is written considering that more
than one amplification reaction will be
performed at the same time. If only one
reaction is planned then there is no need
to prepare the Taq Enzyme Mix (EM).
Gene Link PCR Buffer
1 X PCR Buffer
10 mM Tris-HCl pH 8.3
50 mM KCl
1.5 mM MgCl2
0.001% Gelatin
Yield and Kinetics
The target will be amplified by up to 106 fold in a successful reaction, but the amplification will
usually plateau at 1-10 µg. Thus, 1 pg of target sequence in the reaction is a good place to begin.
PCR reactions produce product in a nonlinear pattern. Amplification follows a typical exponential
curve until some saturation point is reached. Generally products will not be further amplified once
1-5 µg has been generated. Saturation by one product of a reaction does not always prevent
further amplification of other generally unwanted products. Over-cycling may decrease the quality
of an otherwise good reaction. When first optimizing a reaction, it is advisable to take samples
every 5 or 10 cycles to determine the number of cycles actually needed.
146
PCR Additives
DNA polymerases need to elongate rapidly and accurately to function effectively in vivo and in vitro, yet certain DNA regions
appear to interfere with their progress. One common problem is pause sites, at which DNA polymerase molecules cease
elongation for varying lengths of time. Many strong DNA polymerase pauses are at the beginnings of regions of strong
secondary structure such as template hairpins (1). Taq polymerase used in PCR suffers the same fate and GC-rich DNA
sequences often require laborious work to optimize the amplification assay. The GC-rich sequences possess high thermal and
structural stability, presumably because the high duplex melting temperature that permits stable secondary structures to
form, thus preventing completion of a faithful replication (2).
Nucleotide analog 7-deaza dGTP is effective in reducing the secondary structure associated with GC rich region by reducing
the duplex stability (4). Betaine, DMSO and formamide reduces the Tm and the complex secondary structure, thus the duplex
stability (1-5). Tetramethyl ammonium chloride (TMAC) actually increases the specificity of hybridization and increases the
Tm. The use of TMAC is recommended in PCR conditions using degenerate primers.
These PCR additives and enhancing agents have been used to increase the yield, specificity and consistency of PCR reactions.
These additives may have beneficial effects on some amplification and it is impossible to predict which agents will be useful in
a particular context and therefore they must be empirically tested for each combination of template and primers.
Additive
7-deaza-2'-deoxyguanosine;
7-deaza dGTP
Betaine
(N,N,N-trimethylglycine
=
[carboxymethyl]trimethylammo
nium)
BSA
(bovine serum albumin)
DMSO
(dimethyl sulfoxide)
Formamide
Non-ionic detergents
e.g. Triton X-100, Tween 20 or
Nonidet P-40 (NP-40)
TMAC
(tetramethylammonium
chloride)
PCR Additives
Purpose & Function
GC rich region amplification. Reduce the
stability of duplex DNA
Concentration
Totally replace dGTP with 7-deaza dGTP; or
use 7-deaza dGTP: dGTP at 3:1
Reduces Tm facilitating GC rich region
amplification. Reduces duplex stability
Use 3.5M to 0.1M betaine. Be sure to use
Betaine or Betaine (mono)hydrate and not
Betaine HCl.
BSA has proven particularly useful when
attempting to amplify ancient DNA or
templates, which contain PCR inhibitors
such as melanin.
DMSO is thought to reduce secondary
structure and is particularly useful for GC
rich templates.
BSA concentration of 0.01 µg/µl to 0.1 µg/ µl
can be used.
Reduces secondary structure and is
particularly useful for GC rich templates.
Non-ionic detergents stabilise Taq
polymerase and may also supress the
formation of secondary structure.
TMAC is used to reduce potential DNARNA mismatch and improve the
stringency of hybridization reactions. It
increases Tm and minimizes mis-pairing.
DMSO at 2-10% may be necessary for
amplification of some templates, however
10% DMSO can reduce Taq polymerase
activity by up to 50% so it should not be used
routinely.
Formamide is generally used at 1-5%. Do not
exceed 10%.
0.1-1% Triton X-100, Tween 20 or NP-40
may increase yield but may also increase
non-specific amplification. As little as 0.01%
SDS contamination of the template DNA (leftover from the extraction procedure) can
inhibit PCR by reducing Taq polymerase
activity to as low as 10%, however, inclusion
of 0.5% Tween-20 or -40 will effectively
neutralize this effect.
TMAC is generally used at a final
concentration of 15-100 mM to eliminate nonspecific priming.
● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●
147
Purification of PCR Product
Various purification methods are available for the purification of PCR products. The selection of a particular method over
another is based on the downstream application and the initial robustness of the amplification. Usually no further purification
is required for most cloning experiments if a single fragment is amplified, whereas for sequencing applications the amplified
product should be purified from the primers and any other minor amplification products.
The preferred method of purification of an amplified fragment is the excision of the fragment band after agarose gel
electrophoresis. This method yields the purification of a single fragment; as such care should be taken to excise a gel piece
containing a single electrophoretically resolved fragment. The Omni-Clean™ Purification System available from Gene Link can
be used for this purpose. Catalog No. 40-4110-10 for bead based system; 40-4120-10 for spin column based system and 404130-10 for DNA concentration. Please refer to product insert for detailed protocol or visit www.genelink.com.
A.
Purification of DNA from gel slices using glass beads. Provides purified single fragment.
[Omni-Clean™ Gel DNA Beads Purification System; Catalog No. 40-4110-10]
Protocol
1. By weight, determine the volume of the excised DNA fragment.
2. Add 3 volumes of NaI solution and heat to 55 °C. Visually determine the dissolution of gel pieces.
3. Add 1 µl of glass bead suspension per µg of DNA and vortex.
4. Centrifuge at 2K rpm for 20 seconds to pellet glass bead/DNA complex. Discard supernatant.
5. Re-suspend pellet in 400 µl Omni-Clean™ wash buffer. Centrifuge at 2K rpm for 20 seconds and discard wash buffer.
6. Pipet out any remaining buffer in the tube.
7. Add 25 µl water or TE; re-suspend pellet and centrifuge at 2K rpm for 20 seconds.
8. The supernatant contains the purified DNA. Using a pipet, collect the supernatant and transfer to a new appropriately
labeled tube.
B.
Purification of DNA from gel slices using spin column. Provides purified single fragment.
[Omni-Clean™ Gel DNA Spin Column Purification System; Catalog No. 40-4120-50]
Protocol
1. By weight, determine the volume of the excised DNA fragment.
2. Add 3 volumes of NaI solution and heat to 55 °C. Visually determine the dissolution of gel pieces.
3. Add the above solution to the spin column assembled on a collection tube.
4. Let the solution flow by gravity or centrifuge at 2K rpm for 20 seconds. Discard flow through collected in the
collection tube.
5. Add 400 µl Omni-Clean™ wash buffer to the spin column. Centrifuge at 2K rpm for 2 minutes and discard wash buffer
collected in the collection tube.
6. Replace the collection tube with a new appropriately labeled 1.5ml tube.
7. Add 25 µl water or TE to the spin column. Let sit for 3 minutes.
8. Centrifuge at 2K rpm for 2 minutes.
9. The collection tube contains the purified DNA.
C.
Purification of DNA from solution using glass beads. Provides removal of salts, primers and dNTP.
[Omni-Clean™ DNA Beads Concentration System; Catalog No. 40-4130-10]
Protocol
1. Determine volume of DNA solution and add 3 volumes of NaI solution.
2. Add 1 µl of glass bead suspension per µg of DNA.
3. Centrifuge at 2K rpm for 20 seconds to pellet glass bead/DNA complex. Discard supernatant.
4. Re-suspend pellet in 400 µl Omni-Clean™ wash buffer.
5. Centrifuge at 2K rpm for 20 seconds and discard wash buffer.
6. Pipet out any remaining buffer in the tube.
7. Add 25 µl water or TE; re-suspend pellet and centrifuge at 2K rpm for 20 seconds.
8. The supernatant contains the purified DNA. Using a pipet, collect the supernatant and transfer to a new appropriately
labeled tube.
D.
Purification of DNA from solution using spin column. Provides removal of salts, primers and dNTP.
[Omni-Clean™ DNA Spin Column Concentration System; Catalog No. 40-4140-10]
Protocol
1. Determine volume of DNA solution and add 3 volumes of NaI solution.
2. Add the above solution to the spin column assembled on a collection tube.
3. Let the solution flow by gravity or centrifuge at 2K rpm for 20 seconds. Discard flow through collected in the
collection tube.
4. Add 400 µl Omni-Clean™ wash buffer to the spin column. Centrifuge at 2K rpm for 2 minutes and discard wash buffer
collected in the collection tube.
148
5.
6.
7.
8.
Replace the collection tube with a new appropriately labeled 1.5ml tube.
Add 25 µl water or TE to the spin column. Let sit for 3 minutes.
Centrifuge at 2K rpm for 2 minutes.
The collection tube contains the purified DNA.
PEG Precipitation
Primers and salts are efficiently removed by a simple PEG precipitation. This method is recommended for downstream DNA
sequencing application. This method is generally used for plasmid DNA.
Protocol
1. To 50 µl of amplified PCR reaction add 6.0 µl of 5 M NaCl and 40 µl of 13% (w/v) PEG 8000. Incubate the mixture on
ice for 20-30 minutes.
2. Collect the DNA precipitate by centrifugation at maximum speed for 15 minutes at 4 °C in a microfuge. Carefully
remove the supernatant by gentle aspiration.
The pellet of DNA is translucent and generally invisible at this stage.
3. Rinse the pellet with 500 µl of 70% ethanol.
The precipitate changes to a milky-white color and becomes visible.
4. Carefully pour off the 70% ethanol. Rinse the DNA pellet once more with 70% ethanol. Store the tube in an inverted
position at room temperature until the last visible traces of ethanol have evaporated.
5. Dissolve the DNA in 20 µl of H20.
6. Run an aliquot on an agarose gel to confirm the presence of the correct amplified product. The purified DNA is
sequence grade and can be used directly for sequencing.
Primers and salts are efficiently removed by gel filtration using Sephadex G-50. This method is recommended for downstream
DNA sequencing application.
Protocol
1. Hydrate Sephadex G-50 ahead of time in sterile water or TE (10mM Tris pH 8, 1 mM EDTA). Take out from fridge if
already stored hydrated. Bring to room temperature.
2. Assemble a spin column on a collection tube.
3. Add 700 µl of hydrated Sephadex G-50 to each spin column, initiate flow using rubber bulb or any other method.
4. Allow flowing by gravity till there is no more fluid left above the Sephadex G-50 bed. Discard flow through from the
collection tube.
5. Spin the spin column placed inside the collection tube for 2 minutes at 3 K rpm.
6. Change collection tube to new 1.5 ml tube appropriately labeled with sample name.
7. Apply up to 50 µl sample gently to the G-50 bed of the column.
8. Spin for 2 minutes at 3 K rpm.
9. Purified sample is collected in the collection tube. The eluent collected in the 1.5 ml tube is free of salts and primers
shorter than 35-40mer.
References
1. Kovarova, M; and Draber, P; (2000) New Specificity and yield enhancer for polymerase chain reactions (2000) Nucl. Acids.
Res. 28: e70.
2. Henke, W., Herdel, K., Jung, K., Schnorr, D. and Stefan A. Loening, S. (1997) Betaine improves the PCR amplification of
GC-rich DNA sequences. Nucl. Acids Res. 25: 3957-3958.
3. Daniel S. Mytelka, D.S., and Chamberlin, M.J.,(1996) Analysis and suppression of DNA polymerasepauses associated with a
trinucleotide consensus. Nuc. Acids Res.,. 24:2774–278.
4. Keith, J. M., Cochran, D.A.E., Lala, G.H., Adams, P., Bryant, D.and Mitchelson, K.R. (2004) Unlocking hidden genomic
sequence. Nucl. Acids Res. 32: e35.
5. Owczarzy, R., Dunietz, I., Behlke, M.A., Klotz, I.M. and Joseph A. Walder. (2003) Thermodynamic treatment of
oligonucleotide duplex–simplex equilibria. PNAS, 100:14840-14845.
149
CHEMILUMINESCENT BLOT IMAGING WITH X-RAY FILM
Exposure Time
Exposure of the chemiluminescent blot to X-ray film has to be empirically determined initially for the correct duration. An
initial exposure for 5 minutes reveals the duration length for the next exposure. Gene Link recommends the following initial
exposure time.
5 minutes for PCR-Prober™ product line.
Overnight for Gene-Prober™ product line.
X-Ray Film
Emission of light on blue sensitive X-ray film results in a detectable image upon development. Make sure the film cassettes
and developer trays are clean. Prefer using glass dishes, plastic dishes tend to have slight imperfections that lead to
scratching of membranes. Lay the protected blot target side up in the sheet in the cassette. Film must always be protected
from light. In addition, film is sensitive to temperature, moisture, electrical charge and chemicals. You may want to put a
small notch or bend in the film corner as a reference to orient the blot. Use only red light in the film presence. After exposure,
continue processing the film in the dark. Place the film on the blot and close the cassette and expose for 5 minute or longer.
Always handle the film from the edges with clean, dry hands. For the cleanest films, only handle the film at the edge. Use
clean, dry hands to handle the film. Rinse film with water before and after fixing step.
Gene Link recommends the use of blue sensitive X-ray films. We use Kodak X-OMAT or Fuji Super-RX films.
Film Developer and Fixer
Follow manufacturer's recommendations for dilution of the developer and fixer chemistries.
Gene Link uses Kodak chemistry. The developer is GBX, Sigma #P-7042 and the fixer is GBX, Sigma #P-7167.
We dilute 20 ml of the concentrates to 100 ml with distilled water. Prepare fresh developer and fixer with quality water as
specified on the label. Dissolved minerals or particulate may cause noise or loss of signal. Do not use developer that has
turned brown or fixer that has a slimy feel. The developer will oxidize and turn brown if left exposed to air. If you plan to reuse the chemicals throughout the day or next day, place the prepared chemicals in enclosed bottles between use to minimize
oxidation. Be aware that signal may be sacrificed by using older developing chemistries.
Manual Film Developing
Place film in developer for one minute with gentle shaking. Rinse extensively with water. Place in fixer for 30 seconds with
gentle shaking. Rinse with water for 30 seconds. Make sure not to contaminate the developer with fixer because it will
inactivate the developer. If the developer has been contaminated with fixer, it may have a slimy feel. It will not be able to
generate signal on the exposed film. Insufficient fixing or contaminated fixing solutions will result in poor image development,
film discoloration and image fading.
Developing Machines
Developing machines are very useful for high-volume film users. They use the same chemicals in larger volumes than with
manual processing. The machine lines and tanks must be kept clean and the developing chemistries should be made fresh.
150
Genetic Glossary
151
152
Genetic Glossary
Achondroplasia: The most common and well known form of short limbed dwarfism characterized by a normal trunk size with disproportionally
short arms and legs, and a disproportionally large head; autosomal dominant condition.
Advanced maternal age: Women over age 34 (age 35 at delivery) at increased risk for nondisjunction trisomy in fetus.
Affected: An individual who manifests symptoms of a particular condition.
Alcoholism: A chronic and progressive condition characterized by the inability to control the consumption of alcohol.
Allele: An alternative form of a gene; any one of several mutational forms of a gene.
Allele frequency: (synonym: gene frequency) The proportion of individuals in a population who have inherited a specific gene mutation or
variant.
Allele-specific oligonucleotide testing: (synonyms: ASO, ASO testing) The detection of a specific mutation using a synthetic segment of DNA
approximately 20 base pairs in length (an oligonucleotide) that binds to and hence identifies the complementary sequence in a DNA sample.
Allelic heterogeneity: (synonym: molecular heterogeneity) Different mutations in the same gene at the same chromosomal locus that cause a
single phenotype.
Allelic variant of unknown significance: An alteration in the normal sequence of a gene, the significance of which is unclear until further study of
the genotype and corresponding phenotype in a sufficiently large population; complete gene sequencing often identifies numerous (sometimes
hundreds) allelic variants for a given gene.
Alternate paternity: (synonyms: false paternity, nonpaternity) The situation in which the alleged father of a particular individual is not the
biological father.
Alu repetitive sequence: The most common dispersed repeated DNA sequence in the human genome accounting for 5% of human DNA. The
name is derived from the fact that these sequences are cleaved by the restriction endonuclease Alu.
Amino acid sequence: The linear order of the amino acids in a protein or peptide.
Amniocentesis: Prenatal diagnosis method using cells in the amniotic fluid to determine the number and kind of chromosomes of the fetus and,
when indicated, perform biochemical studies.
Amniocyte: Cells obtained by amniocentesis.
Amplification: Any process by which specific DNA sequences are replicated disproportionately greater than their representation in the parent
molecules.
Analyte: A complex biological component of an enzymatic reaction; a substance that is typically measured in a Biochemical/Metabolic specialty
laboratory that is absent, reduced in quantity, or increased in quantity, as a result of an abnormality in a metabolic pathway.
Alpha-fetoprotein (AFP): A protein excreted by the fetus into the amniotic fluid and from there into the mother's bloodstream through the
placenta.
Aneuploidy: State of having variant chromosome number (too many or too few). (i.e. Down syndrome, Turner syndrome).
Angelman syndrome: A condition characterized by severe mental deficiency, developmental delay and growth deficiency, puppet-like gait and
frequent laughter unconnected to emotions of happiness.
Aneuploidy: The occurrence of one or more extra or missing chromosomes leading to an unbalanced chromosome complement, or, any
chromosome number that is not an exact multiple of the haploid number.
Anticipation: The tendency in certain genetic disorders for individuals in successive generations to present at an earlier age and/or with more
severe manifestations; often observed in disorders resulting from the expression of a trinucleotide repeat mutation that tends to increase in size
and have a more significant effect when passed from one generation to the next.
Apert syndrome: A condition caused by the premature closure of the sutures of the skull bones, resulting in an altered head shape, with webbed
fingers and toes. Autosomal dominant.
Artificial insemination: The placement of sperm into a female reproductive tract or the mixing of male and female gametes by other than
natural means.
Ashkenazi Jewish: (synonym: Eastern European Jewish) The Eastern European Jewish population primarily from Germany, Poland, and Russia,
as opposed to the Sephardic Jewish population primarily from Spain, parts of France, Italy, and North Africa.
Autosomal: Refers to any of the chromosomes other than the sex-determining chromosomes (i.e., the X and Y) or the genes on these
chromosomes.
Autosomal dominant: Describes a trait or disorder in which the phenotype is expressed in those who have inherited only one copy of a
particular gene mutation (heterozygotes); specifically refers to a gene on one of the 22 pairs of autosomes (non-sex chromosomes).
153
Autosomal recessive: Describes a trait or disorder requiring the presence of two copies of a gene mutation at a particular locus in order to
express observable phenotype; specifically refers to genes on one of the 22 pairs of autosomes (non-sex chromosomes).
Autosome: A nuclear chromosome other than the X- and Y-chromosomes.
Autoradiograph: A photographic picture showing the position of radioactive substances in tissues.
Bacteriophage: A virus whose host is a bacterium; commonly called phage.
Barr body: The condensed single X-chromosome seen in the nuclei of somatic cells of female mammals. base pair a pair of hydrogen-bonded
nitrogenous bases (one purine and one pyrimidine) that join the component strands of the DNA double helix.
Base sequence: A partnership of organic bases found in DNA and RNA; adenine forms a base pair with thymine (or uracil) and guanine with
cytosine in a double-stranded nucleic acid molecule.
Baysian analysis: A mathematical method to further refine recurrence risk taking into account other known factors.
Becker muscular dystrophy: X-linked condition characterized by progressive muscle weakness and wasting; manifests later in life with
progression less severe than Duchenne muscular dystrophy.
Carrier: An individual who has a recessive, disease-causing gene mutation at a particular locus on one chromosome of a pair and a normal allele
at that locus on the other chromosome; may also refer to an individual with a balanced chromosome rearrangement.
Carrier rate: (synonym: carrier freqency) The proportion of individuals in a population who have a single copy of a specific recessive gene
mutation.
Carrier testing: (synonyms: carrier detection, heterozygote testing) Testing used to identify usually asymptomatic individuals who have a gene
mutation for an autosomal recessive or X-linked recessive disorder. cDNA: Complementary DNA produced from a RNA template by the action of
RNA- dependent DNA polymerase.
Centimorgan: A unit of genetic distance, between two loci on a chromosome. Symbol, cM. The morgan is not used, only the centimorgan. The
genetic distance between two loci is 1 cM if their statistically corrected recombination frequency is 1%; the genetic distance in centimorgans is
numerically equal to the recombination frequency expressed as a percentage. Typically a genetic distance of 1 cM corresponds to a physical
distance of roughly one million base pairs.The centimorgan is named for Thomas Hunt Morgan (1866–1945), who won a Nobel Prize for his work
on the genetics of fruit flies.
Centromere: A region of a chromosome to which spindle traction fibers attach during mitosis and meiosis; the position of the centromere
determines whether the chromosome is considered an acrocentric, metacentric or telomeric chromosome.
Charcot-Marie Tooth disease: A condition characterized by degeneration of the motor and sensory nerves that control movement and feeling in
the arm below the elbow and the leg below the knee; transmitted in autosomal dominant, autosomal recessive and X-linked forms.
Chorionic villus sampling: An invasive prenatal diagnostic procedure involving removal of villi from the human chorion to obtain chromosomes
and cell products for diagnosis of disorders in the human embryo.
Chromosome: Physical structure consisting of a large DNA molecule organized into genes and supported by proteins called chromatin.
Chromosome banding: A technique for staining chromosomes so that bands appear in a unique pattern particular to the chromosome.
Cis configuration: (synonyms: cis, coupling) Term which indicates that an individual who is heterozygous at two neighboring loci has the two
mutations in question on the same chromosome.
Cleft lip/palate: Congenital condition with cleft lip alone, or with cleft palate; cause is thought to be multifactorial.
Clone: An identical copy of a DNA sequence or entire gene; one or more cells derived from and identical to a single ancestor cell; to isolate a
gene or specific sequence of DNA.
Coding region: (synonym: open reading frame) All exons of a gene that contribute to the protein product(s) of the gene.
Codon: In DNA or RNA, a sequence of three nucleotides that codes for a certain amino acid or signals the termination of translation (stop or
termination codon).
Compound heterozygote: An individual who has two different abnormal alleles at a particular locus, one on each chromosome of a pair; usually
refers to individuals affected with an autosomal recessive disorder.
Conformation-sensitive gel electrophoresis: (synonym: CSGE) A type of mutation scanning in which a segment of DNA is screened for mismatch
pairing between normal and mutated base pairsCodon -- a sequence of three nucleotides in mRNA that specifies an amino acid.
Consanguinity: Genetic relationship. Consanguineous individuals have at least one common ancestor in the preceding few generations.
Congenital: Present from birth, but not necessarily genetic.
Conservative change: An amino acid change that does not affect significantly the function of the protein.
Contiguous genes: Genes physically close on a chromosome that when acting together express a phenotype.
154
Consanguinity: Genetic relatedness between individuals descended from at least one common ancestor.
Cosmids: Plasmid vectors designed for cloning large fragments of eukaryotic DNA; the vector is a plasmid into which phage lambda cohesive
end sites have been inserted.
Consultand: The individual (not necessarily affected) who presents for genetic counseling and through whom a family with an inherited disorder
comes to medical attention.
Contiguous gene syndrome: (synonyms: contiguous gene deletion syndrome, microdeletion syndrome) A constellation of findings caused by a
small chromosome deletion or duplication that spans two or more adjacent genes.
Cosegregation: The inclusion of two or more linked genes on a chromosome in the same gamete leading to their transmission together.
Cornelia de Lange syndrome: Condition involving growth deficiency, significant developmental delay, anomalies of the extremities and a
characteristic facial appearance.
CpG islands: Areas of multiple CG repeats in DNA.
Cri-du-chat syndrome: A chromosomal condition (monosomy 5p). Name comes from the distinctive mewing cry of affected infants;
characterized by significant mental deficiency, low birthweight, failure to thrive and short stature; deletion of a small section of the short arm of
chromosome 5.
Crossing over: (synonym: recombination) The exchange of a segment of DNA between two homologous chromosomes during meiosis leading to
a novel combination of genetic material in the offspring.
Crossovers: The exchange of genetic material between two paired chromosome during meiosis.
Cryptic chromosome translocation: A chromosome translocation or rearrangement detected by special techniques (e.g., fluorescent in situ
hybridization [FISH], telomeric detection) because it is too small to be seen with conventional cytogenetic techniques.
Cystic fibrosis: An autosomal recessive genetic condition of the exocrine glands, which causes the body to produce excessively thick, sticky
mucus that clogs the lungs and pancreas, interfering with breathing and digestion.
Cytogenetics: The study of the structure, function, and abnormalities of human chromosomes de novo mutation: (synonyms: de novo gene
mutation, new gene mutation, new mutation) An alteration in a gene that is present for the first time in one family member as a result of a
mutation in a germ cell (egg or sperm) of one of the parents or in the fertilized egg itself
Degenerate codon: A codon that specifies the same amino acid as another codon.
Deletion: Absence of a segment of DNA; may be as small as a single base or as large as one or more genes
Deletion mapping: The use of overlapping deletions to localize the position of an unknown gene on a chromosome or linkage map.
Denaturing gradient gel electrophoresis: (synonym: DGGE) Identification of mutations by electrophoresis of double-stranded DNA samples
through a denaturing gradient, such as urea. Certain mutations affect the migration pattern by changing the point in the gel at which the DNA
denatures; mutant sequences can be distinguished from wild-type sequences by comparing the electrophoretic pattern.
Densitometry: Method of identifying gene dosage or expression by measurement of light absorption on an autoradiogram (film) of a band (or
spot) representing a DNA, RNA, or protein sample; useful in detecting duplication mutations and heterozygous deletion mutations
Derivative chromosome: Term used to denote an abnormal chromosome consisting of segments from two or more chromosomes joined
together as the result of a translocation, insertion, or other rearrangement
DGGE: (synonym: denaturing gradient gel electrophoresis) Identification of mutations by electrophoresis of double-stranded DNA samples
through a denaturing gradient, such as urea. Certain mutations affect the migration pattern by changing the point in the gel at which the DNA
denatures; mutant sequences can be distinguished from wild-type sequences by comparing the electrophoretic pattern.
Diagnostic testing: Testing designed to confirm or exclude a known or suspected genetic disorder in a symptomatic individual or, prenatally, in
a fetus at risk for a certain genetic condition
Diploid: The normal number of chromosomes in a somatic cell; in humans, 46 chromosomes (22 pairs of autosomes and two sex
chromosomes).
Direct DNA analysis: (synonym: direct DNA) The use of mutation analysis, mutation scanning, sequence analysis, or other means of molecular
genetic testing to detect a genetic alteration associated with a specific disorder; direct DNA analysis is possible only when the gene (or genes)
or genomic region associated with a disorder is known
Disease-causing mutation: A gene alteration that causes or predisposes an individual to a specific disease.
DMD: Duchenne muscular dystrophy.
DNA: (synonym: Deoxyribonucleic acid) The molecule which encodes the genes responsible for the structure and function of an organism and
allows for transmission of genetic information from one generation to the next Disease: Any deviation from the normal structure or function of
any part, organ, or system of the body that is manifested by a characteristic set of symptoms and signs whose pathology and prognosis may be
known or unknown.
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DNA banking: The process through which DNA is extracted from any of a number of possible cell sources and stored indefinitely by freezing or
refrigerating for future testing; done when a specific test is not presently available or when the decision to have testing has not been made
DNA-based testing: (synonyms: DNA testing, molecular genetic testing) Testing that involves the analysis of DNA, either through linkage
analysis, sequencing, or one of several methods of mutation detection.
DNA hybridization: A technique for selectively binding specific segments of single-stranded (ss) DNA or RNA by base pairing to complementary
sequences on ssDNA molecules that are trapped on a nitrocellulose filter.
DNA probe: Any biochemical used to identify or isolate a gene, a gene product, or a protein.
DNA sequencing: "Plus and minus" or "primed synthesis" method, developed by Sanger, DNA is synthesized in vitro in such a way that it is
radioactively labeled and the reaction terminates specifically at the position corresponding to a given base; the "chemical" method, ssDNA is
subjected to several chemical cleavage protocols that selectively make breaks on one side of a particular base.
Domain: A specific region or amino acid sequence in a protein associated with a particular function or corresponding segment of DNA.
Dominant: Alleles that determine the phenotype displayed in a heterozygote with another (recessive) allele.
Dominant negative mutation: A mutation whose gene product adversely affects the normal, wild-type gene product within the same cell, usually
by dimerizing (combining) with it. In cases of polymeric molecules, such as collagen, dominant negative mutations are often more deleterious
than mutations causing the production of no gene product (null mutations or null alleles).
Dosage analysis: Method of measuring the quantity of a variety of analytes, including DNA, RNA, and protein, by comparison with a known
standard; can be used to determine the number of copies of a sequence of DNA (i.e., to test for duplication and deletion mutations) either by
visual comparison of band intensity or numerical quantification by densitometry. If extra copies of a gene are present, intensity is greater than
100% on a gel or film; whereas, if one copy of the gene is missing, the intensity is approximately 50%.
Double heterozygote: An individual who is heterozygous for a mutation at each of two separate genetic loci.
Down syndrome: A type of mental deficiency due to trisomy (three copies) of autosome 21, a translocation of 21 or mosaicism.
Duchenne/Becker muscular dystrophy: The most common and severe form of muscular dystrophy; transmitted as an X-linked trait. X-linked
recessive. Symptoms include onset at 2-5 years with difficulty with gait and stairs, enlarged calf muscles, progression to wheelchair by
adolescence, shortened life span.
Duplication: The presence of an extra segment of DNA, resulting in redundant copies of a portion of a gene, an entire gene, or a series of
genes, usually caused by unequal crossing-over during gene replication when gametes are formed in meiosis.
Dysmorphology: The clinical study of malformation syndromes.
Dystonia: Neurologic condition involving repeated twisting and movement. Involves a variety of muscle groups. Intelligence not effected. Three
forms: childhood - autosomal dominant, autosomal recessive, adult-acquired.
Dwarfism: Conditions of short stature with adult height under 4'10" as adult, usually with normal intelligence and lifespan. Ehlers Danlos
Syndrome connective tissue condition including problems with tendons, ligaments, skin, bones, cartilage, and membranes surrounding blood
vessels and nerves. Symptoms include joint laxity, elastic skin, dislocations. Many forms: autosomal dominant, autosomal recessive, X-linked
forms.
ELSI: Ethical, legal and social implications (of HGP).
Endonuclease: An enzyme that breaks the internal phosphodiester bonds in a DNA molecule.
Enzyme assay: Measurement of enzyme activity with a particular substrate; can be assessed in a variety of ways including quantification of the
end product or colorimetric analysis.
Erythrocytes: The hemoglobin-containing cell found in the blood of vertebrates.
Euchromatin: The chromatin that shows the staining behavior characteristic of the majority of the chromosomal complement.
Eugenics: The improvement of humanity by altering its genetic composition by encouraging breeding of those presumed to have desirable
genes.
Euploid: Any chromosome number that is a multiple of the haploid number.
Exon: Coding sequence of DNA present in mature messenger RNA; DNA initially transcribed to messenger RNA consists of coding sequences
(exons) and non-coding sequences (introns). Introns are spliced out of the messenger RNA prior to translation, leaving only the exons to
ultimately encode the amino acid product.
Exons: Portion of a gene included in the transcript of a gene and survives processing of the RNA in the cell nucleus to become part of a spliced
messenger of a structural RNA in the cell cytoplasm; an exon specifies the amino acid sequence of a portion of the complete polypeptide.
Exon scanning: The process by which certain exons (coding regions within a gene), under highest suspicion to contain a specific mutation, are
subjected to testing via conformation sensitive gel electrophoresis (CSGE), single-stranded conformational-polymorphism (SSCP), denaturing
gradient gel electrophoresis (DGGE), or other means deemed most appropriate, to confirm the presence of a mutation before use of further
testing, such as sequencing, to delineate the exact nature of the mutation; used to expedite analysis when the disorder in question can be
caused by numerous possible mutations within a gene.
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Ethics: The study of fundamental principles which defines values and determines moral duty and obligation.
False negative result: A test result which indicates that an individual is unaffected and/or does not have a particular gene mutation when he or
she is actually affected and/or does have a gene mutation; i.e., a negative test result in an affected individual.
False paternity: (synonyms: alternate paternity, nonpaternity) The situation in which the alleged father of a particular individual is not the
biological father.
False positive result: A test result which indicates that an individual is affected and/or has a certain gene mutation when he or she is actually
unaffected and/or does not have the mutation; i.e., a positive test result in a truly unaffected individual.
Familial: A phenotype that occurs in more than one family member; may have genetic or non-genetic etiology.
Family history: The genetic relationships and medical history of a family; when represented in diagram form using standardized symbols and
terminology, usually referred to as a pedigree.
Family-specific mutation: In a family, the sequence alteration observed that causes or predisposes to a particular disease; the mutation may be
rare or common.
Fetal alcohol syndrome: A link between excessive alcohol consumption during pregnancy and birth defects; characteristics include small head
and eyes, folds of the skin that obscure the inner juncture of the eyelids, short, upturned nose, and thin lips.
First-degree relative: Any relative who is one meiosis away from a particular individual in a family (i.e., parent, sibling, offspring).
FISH: (synonym: fluorescent in situ hybridization) A technique used to identify the presence of specific chromosomes or chromosomal regions
through hybridization (attachment) of fluorescently-labeled DNA probes to denatured chromosomal DNA. Examination under fluorescent lighting
detects the presence of the hybridized fluorescent signal (and hence presence of the chromosome material) or absence of the hybridized
fluorescent signal (and hence absence of the chromosome material).
FISH-interphase: A technique used to identify the presence of specific chromosomes or chromosomal regions through hybridization of
fluorescent labeled DNA probes to denatured chromosomal DNA. Examination under fluorescent lighting detects the presence of the hybridized
fluorescent signal (and hence presence of the chromosome material) or absence of the hybridized fluorescent signal (and hence absence of the
chromosome material). With interphase FISH, probes are introduced directly to the interphase cell. Interphase FISH is often used for rapid
detection of specific types of aneuploidy in fetal cells and for the detection of certain deletions, duplications and other abnormalities in tumor
cells. In contract to metaphase FISH, interphase FISH does not permit visualization of the actual chromosomes; therefore, certain structural
rearrangements or aneuploidy will not be detected.
FISH-metaphase: A technique used to identify the presence of specific chromosomes or chromosomal regions through hybridization of
fluorescent labeled DNA probes to denatured chromosomal DNA. Examination under fluorescent lighting detects the presence of the hybridized
fluorescent signal (and hence presence of the chromosome material) or absence of the hybridized fluorescent signal (and hence absence of the
chromosome material). With metaphase FISH, cells progress through the division process until metaphase, when chromosomes are condensed
and can be individually distinguished. In contrast to interphase FISH, metaphase FISH permits visualization of the actual chromosomes as well
as the general location of the abnormality on the chromosome.
5' – end: The end of a polynucleotide with a free (or phosphorylated or capped) 5' - hydroxyl group; transcription/translation begins at this end.
Flanking marker: An identifiable, polymorphic region of DNA (i.e., marker) located to the side of a gene (i.e., flanking), as opposed to an
intragenic marker which is located within the gene itself. Flanking markers are used in linkage analysis to track the coinheritance of the gene in
question.
Flanking microsatellite analysis: The use of highly variable repetitive sequences found in microsatellite regions adjacent to genes or other areas
of interest as markers for linkage analysis, DNA fingerprinting, or other diagnostic application
Fluorescent in situ hybridization: (synonym: FISH) A technique used to identify the presence of specific chromosomes or chromosomal regions
through hybridization (attachment) of fluorescently-labeled DNA probes to denatured chromosomal DNA. Examination under fluorescent lighting
detects the presence of the hybridized fluorescent signal (and hence presence of the chromosome material) or absence of the hybridized
fluorescent signal (and hence absence of the chromosome material).
Founder effect: A gene mutation observed in high frequency in a specific population due to the presence of that gene mutation in a single
ancestor or small number of ancestors
Frameshift mutation: (synonyms: out-of-frame deletion, out-of-frame mutation) An insertion or deletion involving a number of base pairs that
is not a multiple of three and consequently disrupts the triplet reading frame, usually leading to the creation of a premature termination (stop)
codon and resulting in a truncated protein product.
Fragile sites: A non-staining gap of variable width that usually involves both chromatids and is always at exactly the same point on a specific
chromosome derived from an individual or kindred.
Fragile-X syndrome: X-linked trait; the second most common identifiable cause of genetic mental deficiency.
Gamete: An haploid cell.gel electrophoresis the process by which nucleic acids (DNA or RNA) or proteins are separated by size according to
movement of the charged molecules in an electrical field.
Gametogenesis: (synonyms: oogenesis, spermatogenesis) The meiotic process by which mature gametes (ova and sperm) are formed.
Oogenesis refers specifically to the production of ova and spermatogenesis to the production of sperm.
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Gene: The basic unit of heredity, consisting of a segment of DNA arranged in a linear manner along a chromosome. A gene codes for a specific
protein or segment of protein leading to a particular characteristic or function.
Gene amplification: Any process by which specific DNA sequences are replicated disproportionately greater than their representation in the
parent molecules; during development, some genes become amplified in specific tissues.
Gene conversion: The transfer of DNA sequences between two homologous genes, most often by unequal crossing over during meiosis; can be
a mechanism for mutation if the transfer of material disrupts the coding sequence of the gene or if the transferred material itself contains one
or more mutations.
Gene product: Genes are transcribed into segments of RNA (ribonucleic acid), which are translated into proteins. Both RNA and proteins are
products of the expression of the gene.
Gene symbol: A unique abbreviation of a gene name consisting of italicized uppercase Latin letters and Arabic numbers formally assigned by the
by HUGO Gene Nomenclature Committee after a gene has been identified (Note: a putative gene may be referred to by its locus name prior to
its identification).
Gene therapy: Experimental treatment of a genetic disorder by replacing, supplementing, or manipulating the expression of abnormal genes
with normally functioning genes.
Gene transfer: The transfer of genetic material, ranging from a small segment of DNA to the entire genome, from a human cell to another type
of cell in culture in order to study the frequency with which known genetic markers are transferred together to the recipient genome; used to
determine the physical proximity of genetic markers in the human genome; also used to study gene expression and regulation.
Genetic counseling: A process, involving an individual or family, comprising: evaluation to confirm, diagnose, or exclude a genetic condition,
malformation syndrome, or isolated birth defect; discussion of natural history and the role of heredity; identification of medical management
issues; calculation and communication of genetic risks; provision of or referral for psychosocial support.
Genetic linkage map: A chromosome map showing the relative positions of the known genes on the chromosomes of a given species.
Genetic screening: Testing groups of individuals to identify defective genes capable of causing hereditary conditions.
Genetic variation: A phenotypic variance of a trait in a population attributed to genetic heterogeneity.
Genetic predisposition: (synonym: genetic susceptibility) Increased susceptibility to a particular disease due to the presence of one or more
gene mutations associated with an increased risk for the disease and/or a family history that indicates an increased risk for the disease.
Genome: The complete DNA sequence, containing all genetic information and supporting proteins, in the chromosomes of an individual or
species. Gene map: The linear arrangement of mutable sites on a chromosome as deduced from genetic recombination experiments.
Genotype: The genetic constitution of an organism or cell; also refers to the specific set of alleles inherited at a locus.
Genotype-phenotype correlation: The association between the presence of a certain mutation or mutations (genotype) and the resulting pattern
of abnormalities (phenotype).
Genotyping: Testing that reveals the specific alleles inherited by an individual; particularly useful for situations in which more than one
genotypic combination can produce the same clinical presentation, as in the ABO blood group, where both the AO and AA genotypes yield type
A blood.
Germ cell: A sex cell or gamete (egg or spermatozoan).Haldane equation Haldane's law: the generalization that if first generation hybrids are
produced between two species, but one sex is absent, rare, or sterile, that sex is the heterogamic sex.
Germline: The cell line from which egg or sperm cells (gametes) are derived
Germline mosaicism: Two or more genetic or cytogenetic cell lines confined to the precursor (germline) cells of the egg or sperm; formerly
called gonadal mosaicism
Germline mutation: The presence of an altered gene within the egg or sperm (germ cell), such that the altered gene can be passed to
subsequent generations
Gonadal mosaicism: See germline mosaicism.
Haploid: Half the diploid or normal number of chromosomes in a somatic cell; the number of chromosomes in a gamete (egg or sperm) cell,
which in humans is 23 chromosomes, one chromosome from each chromosome pair
Haploinsufficiency: The situation in which an individual who is heterozygous for a certain gene mutation or hemizygous at a particular locus,
often due to a deletion of the corresponding allele, is clinically affected because a single copy of the normal gene is incapable of providing
sufficient protein production as to assure normal function.
Haplotype analysis: Molecular genetic testing to identify a set of closely linked segments of DNA; used in linkage analysis or when a given trait
is in linkage disequilibrium with a marker or set of markers.
Hardy-Weinberg Law: The concept that both gene frequencies and genotype frequencies will remain constant from generation to generation in
an infinitely large, interbreeding population in which mating is at random and there is no selection, migration or mutation.
Hemizygous: The situation in which an individual has only one member of a chromosome pair or chromosome segment rather than the usual
two; refers in particular to X-linked genes in males who under normal circumstances have only one X chromosome.
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Hemophilia: A sex-linked disease in humans in which the blood-clotting process is defective.
Heterogeneity: The production of identical or similar phenotypes by different genetic mechanisms.
Heteroplasmy: The situation in which, within a single cell, there is a mixture of mitochondria (energy producing cytoplasmic organelles), some
containing mutant DNA and some containing normal DNA.
Heterozygote: An individual who has two different alleles at a particular locus, one on each chromosome of a pair; one allele is usually normal
and the other abnormal
HGP: Human Genome Project.
HHMI: Howard Hughes Medical Institute.
High-resolution chromosome studies: Analysis of the number and structure of the chromosomes when cell division has been arrested and the
chromosomes stained at an early stage (pro-metaphase) of mitosis. The chromosomes of a high resolution study appear longer and reveal 7001200 bands, allowing more detailed analysis of the chromosome structure, as opposed to the typical 300-600 bands observed with routine
metaphase banding.
Homologous chromosomes: (synonym: homologs) The two chromosomes from a particular pair, normally one inherited from the mother and
one from the father, containing the same genetic loci in the same order.
Homozygote: An individual who has two identical alleles at a particular locus, one on each chromosome of a pair.
Hotspot mutation region: DNA sequences of high susceptibility to mutation due to some inherent instability, tendency toward unequal crossing
over, or chemical predisposition to single nucleotide substitutions; region where mutations are observed with greater frequency.
Homologous chromosomes -- chromosomes that pair during meiosis; each homologue is a duplicate of one chromosome from each parent.
Housekeeping genes: Those genes expressed in all cells because they provide functions needed for sustenance of all cell types.
Huntington disease: A disease characterized by irregular, spasmodic involuntary movements of the limbs and facial muscles, mental
deterioration and death, usually within 20 years of the onset of symptoms. A CAG triple repeat disorder.
Hybridization: The pairing of a single-stranded, labeled probe (usually DNA) to its complementary sequence.
Ichthyosis: Any of several hereditary or congenital skin conditions; skin of affected individuals has a dry, scaly appearance.
Imprinting: The process by which maternally and paternally derived chromosomes are uniquely chemically modified leading to different
expression of a certain gene or genes on those chromosomes depending on their parental origin.
Incomplete penetrance -- the gene for a condition is present, but not obviously expressed in all individuals in a family with the gene.
In-frame mutation: A mutation that does not cause a shift in the triplet reading frame; such mutations can, however, lead to the synthesis of
an abnormal protein product.
Informativeness: In linkage analysis, the ability to distinguish between maternally-inherited and paternally-inherited DNA markers
(polymorphisms) within or near a given gene of interest
Informed consent: Permission given by an individual to proceed with a specific test or procedure, with an understanding of the risks, benefits,
limitations, and potential implications of the procedure itself and its results
Insertion: A chromosome abnormality in which material from one chromosome is inserted into another nonhomologous chromosome; a
mutation in which a segment of DNA is inserted into a gene or other segment of DNA, potentially disrupting the coding sequence
In situ hybridization: Hybridization of a labeled probe to its complementary sequence within intact, banded chromosomes.
Interfamilial variability: Variability in clinical presentation of a particular disorder among affected individuals from different families
Intermediate allele: (synonym: premutation) In disorders caused by trinucleotide repeat expansions, an abnormally large allele that is not
associated with clinical symptoms but that can expand into a full mutation when transmitted to offspring (full mutations are associated with
clinical symptoms of the disorder)
Intrafamilial variability: Variability in clinical presentation of a particular disorder among affected individuals within the same immediate or
extended family
Intragenic marker: An identifiable, polymorphic region of DNA (i.e., marker) located within a gene (i.e., intragenic), as opposed to a flanking
marker, which is located on either side of a gene. Intragenic markers are used in linkage analysis to track the coinheritance of the gene in
question.
Intron: Non-coding sequence of DNA removed from mature messenger RNA prior to translation. DNA initially transcribed to messenger RNA
consists of coding sequences (exons) and non-coding sequences (introns); introns are spliced out of the messenger RNA prior to translation,
leaving only the exons to ultimately encode the amino acid product.
Introns: A segment of DNA (between exons) that is transcribed into nuclear RNA, but are removed in the subsequent processing into mRNA.
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Intronic mutation: A mutation (usually a base substitution) within an intron that creates an alternative splice site that competes with the normal
splice sites during RNA processing. Such a mutation results in a proportion of mature messenger RNA with improperly spliced intron sequences.
Inversion: A chromosomal rearrangement in which a segment of a chromosome has inverted from end to end, and re-inserted into the
chromosome at the same breakage site. Balanced inversions (in which no net loss or gain of genetic material occurs) are usually not associated
with phenotypic abnormalities, however, in some cases, gene disruptions at the breakpoints can cause adverse phenotypic effects, including
some known genetic diseases. Unbalanced inversions (in which loss or gain of chromosome material occurs) nearly always yield an abnormal
phenotype.
Isochromosome: A metacentric chromosome produced during mitosis or meiosis when the centromere splits transversely instead of
longitudinally; the arms of such chromosome are equal in length and genetically identical, however, the loci are positioned in reverse sequence
in the two arms.
Isoelectric focusing: Method of mutation detection by which proteins are separated according to the pH at which their net charge is zero
(isoelectric point); often used in conjunction with a western blot to allow identification of wild-type versus mutant protein products. A DNA
sequence alteration resulting in an amino acid substitution can change the isoelectric point of a protein.
Isoforms: The protein products of different versions of messenger RNA created from the same gene by employing different promoters, which
causes transcription to skip certain exons. Since the promoters are tissue-specific, different tissues express different protein products of the
same gene.
Isolated: An abnormality that occurs in the absence of other systemic involvement.
Karyotype: A photographic representation of the chromosomes of a single cell, cut and arranged in pairs based on their banding pattern and
size according to a standard classification
Kindred: An extended family; term often used in linkage studies to refer to large families.
Klinefelter syndrome: An endocrine condition caused by a an extra X-chromosome (47,XXY); characterized by the lack of normal sexual
development and testosterone, leading to infertility and adjustment problems if not detected and treated early.
Lligase: An enzyme that functions in DNA repair.
Linkage: The greater association in inheritance of two or more nonallelic genes than is to be expected from independent assortment; genes are
linked because they reside on the same chromosome.
Linkage analysis: (synonym: indirect DNA analysis) Testing DNA sequence polymorphisms (normal variants) that are near or within a gene of
interest to track within a family the inheritance of a disease-causing mutation in a given gene.
Linkage disequilibrium: In a population, co-occurrence of a specific DNA marker and a disease at a higher frequency than would be predicted by
random chance.
Locus: The physical site or location of a specific gene on a chromosome.
Locus heterogeneity: The situation in which mutations in genes at different chromosomal loci cause the same phenotype
Locus name: An informally assigned abbreviation used in the process of mapping to designate a putative gene prior to gene identification; once
the gene is identified. The locus name is generally replaced by a formally assigned gene symbol (which often differs from the locus name).
Lod score: Logarithm of the odd score; a measure of the likelihood of two loci being within a measurable distance of each other.
Loss of heterozygosity: (synonym: LOH) At a particular locus heterozygous for a deleterious mutant allele and a normal allele, a deletion or
other mutational event within the normal allele renders the cell either hemizygous (one deleterious allele and one deleted allele) or homozygous
for the deleterious allele
Lyonization: (synonym: X-chromosome inactivation) The phenomenon in females by which one X chromosome (either maternally derived or
paternally derived) is randomly inactivated in early embryonic cells, with fixed inactivation in all descendant cells; first described by the
geneticist Mary Lyon.
Manifesting carrier: An individual who has, at a particular locus, a recessive, disease-causing allele on one chromosome and a normal allele on
the other chromosome and who manifests some symptoms of the disorder; generally refers to female carriers of an X-linked recessive mutation
who are clinically affected, although the phenotype is usually less severe as compared to males with the same mutation.
Mapped gene: (synonym: mapped phenotype) A gene or phenotype whose relative position on a segment of DNA or on a chromosome has been
established.
Marfan syndrome: Autosomal dominant condition of connective tissue; affects the skeletal, ocular and cardiovascular systems.
Marker: An identifiable segment of DNA (e.g., RFLP, VNTR, microsatellite) with enough variation between individuals that its inheritance and coinheritance with alleles of a given gene can be traced; used in linkage analysis.
Marker chromosome: A small chromosome containing a centromere occasionally seen in tissue culture, often in a mosaic state (present in some
cells but not in others). A marker chromosome may be of little clinical significance or, if it contains material from one or both arms of another
chromosome, may create an imbalance for whatever genes are present; assessment to establish clinical significance, particularly if found in a
fetal karyotype, is often difficult.
Maternal contamination: The situation which occurs in prenatal testing in which a sample of chorionic villus, amniotic fluid, or umbilical blood
becomes contaminated with maternal (usually blood) cells, which can confound interpretation of the results of genetic analysis.
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Meiosis: The doubling of gametic chromosome number.
Methylation: The attachment of methyl groups to DNA at cytosine bases; correlated with reduced transcription of the gene and thought to be
the principal mechanism in X-chromosome inactivation and imprinting.
Methylation analysis: Testing that evaluates the methylation status of a gene (attachment of methyl groups to DNA cytosine bases); genes that
are methyalted are not expressed; methylation plays a role in X-chromosome inactivation and imprinting.
Methylmalonic acidemia -- a group of conditions characterized by the inability to metabolize methylmalonic acid or by a defect in the
metabolism of Vitamin B12.
Microdeletion syndrome: (synonym: contiguous gene deletion syndrome) A syndrome caused by a chromosomal deletion spanning several
genes that is too small to be detected under the microscope using conventional cytogenetic methods. Depending on the size of the deletion,
other techniques, such as FISH or other methods of DNA analysis can sometimes be employed to identify the deletion.
Microsatellite: (synonyms: satellite DNA, short tandem repeats) Repetitive segments of DNA two to five nucleotides in length
(dinucleotide/trinucleotide/tetranucleotide/pentanucleotid e repeats), scattered throughout the genome in non-coding regions between genes or
within genes (introns), often used as markers for linkage analysis because of the naturally occurring high variability in repeat number between
individuals. These regions are inherently unstable and susceptible to mutations.
Microsatellite instability: (synonyms: MSI, replication error phenotype, RER) The presence of a discrepancy between the size of microsatellites
in DNA from tumor tissue compared to nontumor tissue from the same person, resulting from mutations in a gene in the DNA mismatch repair
pathway (MMR) that would normally correct these errors.
Mismatch repair mechanism: (synonym: mismatch repair) The DNA 'proof-reading' system controlled by certain genes that identifies, excises,
and corrects errors in the pairing of the bases during DNA replication. Mutations in the genes responsible for this mechanism can lead to certain
genetic diseases and some forms of cancer.
Missense mutation: A single base pair substitution that results in the translation of a different amino acid at that position.
Mitochondrial DNA: The mitochondrial genome consists of a circular DNA duplex, with 5 to 10 copies per organelle.
Mitochondrial inheritance: Mitochondria, cytoplasmic organelles that produce the energy source ATP for most chemical reactions in the body,
contain their own distinct genome; mutations in mitochondrial genes are responsible for several recognized syndromes and are always
maternally inherited since ova contain mitochondria, whereas sperm do not.
Mitosis: Nuclear division.
Mode of inheritance: (synonyms: inheritance pattern, pattern of inheritance) The manner in which a particular genetic trait or disorder is passed
from one generation to the next. Autosomal dominant, autosomal recessive, X-linked dominant, X-linked recessive, multifactorial, and
mitrochondrial inheritance are examples.
Molecular genetic testing: (synonyms: DNA testing, DNA-based testing, molecular testing) Testing that involves the analysis of DNA, either
through linkage analysis, sequencing, or one of several methods of mutation detection.
Monosomy: The presence of only one chromosome from a pair; partial monosomy refers to the presence of only one copy of a segment of a
chromosome.
Mosaicism: Within a single individual or tissue, the occurrence of two or more cell lines with different genetic or chromosomal constitutions.
mRNA: Messenger RNA; an RNA molecular that functions during translation to specify the sequence of amino acids in a nascent polypeptide.
Multifactorial: A characteristic influenced in its expression by many factors, both genetic and environmental.
Multifactorial inheritance: (synonym: polygenic) The combined contribution of one or more often unspecified genes and environmental factors,
often unknown, in the causation of a particular trait or disease.
Mutation: (synonyms: sequence alteration, splicing mutation) Any alteration in a gene from its natural state; may be benign (commonly
referred to as a "polymorphism"), pathogenic, or of unknown significance.
Mutation analysis: Testing for the presence of a specific mutation (e.g., Glu6Val for sickle cell anemia), a specific type of mutation (e.g., the
trinucleotide repeat expansion associated with spinocerebellar ataxia type 1, deletions associated with Duchenne muscular dystropy), or set of
mutations (e.g., a panel of mutations for cystic fibrosis), as opposed to complete gene sequencing or mutation scanning, which detect most
mutations in the tested region.
Mutation scanning: A process by which a segment of DNA is screened via one of a variety of methods to identify variant gene region(s). Variant
regions are further analyzed (by sequence analysis or mutation analysis) to identify the sequence alteration.
Myotonic dystrophy: A combination of progressive weakening of the muscles and muscle spasms or rigidity, with difficulty relaxing a contracted
muscle; inherited as an autosomal dominant trait. A CTG triple repeat disorder.
Negative predictive value: The likelihood that an individual with a negative test result is actually unaffected and/or does not have the particular
gene mutation in question.
Neurofibromatosis: One of the most common single gene conditions affecting the human nervous system; in most cases, "cafe au lait" spots,
are the only symptom; inherited as an autosomal dominant trait, with 50% being new mutations.
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Newborn screening: Testing done within days of birth to identify infants at increased risk for a specific genetic disorder so that treatment can
begin as soon as possible; when a newborn screening result is positive, further diagnostic testing is usually required to confirm or specify the
results and counseling is offered to educate the parents.
Nonsense mutation: A single base pair substitution that prematurely codes for a stop in amino acid translation (stop codon).
Noonan syndrome: A condition characterized by short stature and ovarian or testicular dysfunction, mental deficiency, and lesions of the heart.
Northern analysis: A technique for transferring electrophoretically resolved RNA segments from an agarose gel to a nitrocellulose filter paper
sheet via capillary action.
Northern blot: (synonym: northern blotting analysis) The separation of sequences or fragments of RNA, partially digested by endonucleases, on
an electrophoretic gel.
Novel mutation: A distinct gene alteration that has been newly discovered; not the same as a 'new' or 'de novo' mutation.
Nucleotide: A molecule consisting of a nitrogenous base (adenine, guanine, thymine, or cytosine in DNA; adenine, guanine, uracil, or cytosine in
RNA), a phosphate group, and a sugar (deoxyribose in DNA; ribose in RNA). DNA and RNA are polymers of many nucleotides.
Null allele: A mutation that results in either no gene product or the absence of function at the phenotypic level.
Obligate carrier: (synonym: obligate heterozygote) An individual who may be clinically unaffected but who must carry a gene mutation based
on analysis of the family history; usually applies to disorders inherited in an autosomal recessive and X-linked recessive manner.
Obligate heterozygote: (synonym: obligate heterozygote) An individual who may be clinically unaffected but who must carry a gene mutation
based on analysis of the family history; usually applies to disorders inherited in an autosomal recessive and X-linked recessive manner.
Oligonucleotide: (synonym: Oligo) A short stretch of DNA; usually 15 to 250 nucleotide long. The word oligo is used interchangeably with
“primers” used for PCR or sequencing.
Open reading frame: (synonym: ORF) All exons of a gene that contribute to the protein product(s) of the gene. Oncogenes: Genes involved in
cell cycle control (growth factors, growth factor regulator genes, etc), a mutation can lead to tumor growth.
Osteogenesis imperfecta: A condition also known as brittle bone disease; characterized by a triangular shaped face with yellowish brown teeth,
short stature and stunted growth, scoliosis, high pitched voice, excessive sweating and loose joints.
Paracentric inversion: An inversion in which the breakpoints are confined to one arm of a chromosome; the inverted segment does not span the
centromere.
Parent-of-origin studies: An analysis used to determine whether a particular chromosome or segment of DNA was inherited from an individual's
mother or father; helpful in the diagnosis of disorders in which imprinting or uniparental disomy is a possible underlying etiological mechanism.
Parentage testing: (synonyms: maternity testing, paternity testing) The process through which DNA sequences from a particular child and a
particular adult are compared to estimate the likelihood that the two individuals are related; DNA testing can reliably exclude but cannot
absolutely confirm an individual as a biological parent.
Parthenogenesis: The development of an individual from an egg without fertilization.
PCR: (synonym: polymerase chain reaction) A procedure that produces millions of copies of a short segment of DNA through repeated cycles of:
1) denaturation, 2) annealing, and 3) elongation; PCR is a very common procedure in molecular genetic testing and may be used to: 1)
generate a sufficient quantity of DNA to perform a test (e.g., sequence analysis, mutation scanning), or 2) may be a test in and of itself (e.g.,
allele-specific amplification, trinucleotide repeat quantification).
Pedigree: A diagram of the genetic relationships and medical history of a family using standard symbols and terminology
Penetrance: The proportion of individuals with a mutation causing a particular disorder who exhibit clinical symptoms of that disorder; most
often refers to autosomal dominant conditions.
Pericentric inversion: An inversion in which the breakpoints occur on both arms of a chromosome. The inverted segment spans the centromere.
Phenotype: The observable physical and/or biochemical characteristics of the expression of a gene; the clinical presentation of an individual
with a particular genotype
Physical map: Map where the distance between markers is the actual distance, such as the number of base pairs.
Phenotyping: Diagnostic testing and inference of a particular genotype based on clinical or biochemical presentation (phenotype) of the
individual, such as measurement of alpha-1-antitrypsin level, which is greatly reduced in individuals homozygous for the Z allele. With the
advent of DNA-based testing, direct mutation analysis (genotyping) is becoming more widely available for many disorders.
PKU: Phenylketonuria, an enzyme deficiency condition characterized by the inability to convert one amino acid, phenylalanine, to another,
tyrosine, resulting in mental deficiency. plasmid double-stranded, circular, bacterial DNA into which a fragment of DNA from another organism
can be inserted.
Pleiotropy: The phenomenon of variable phenotypes for a number of distinct and seemingly unrelated phenotypic effects.
Point mutation: An alteration in DNA sequence caused by a single nucleotide base change, insertion, or deletion.
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Polycystic kidney disease (PKD): A group of conditions characterized by fluid filled sacs that slowly develop in both kidneys, eventually resulting
in kidney malfunction.
Polygenic: A condition caused by the additive contributions of mutations in multiple genes at different loci.
Polymerase: Any enzyme that catalyzes the formation of DNA or RNA from deoxyribonucleotides or ribonucleotides.
Polymerase chain reaction (PCR): A procedure that produces millions of copies of a short segment of DNA through repeated cycles of: 1)
denaturation, 2) annealing, and 3) elongation; PCR is a very common procedure in molecular genetic testing and may be used to: 1) generate a
sufficient quantity of DNA to perform a test (e.g., sequence analysis, mutation scanning), or 2) may be a test in and of itself (e.g., allelespecific amplification, trinucleotide repeat quantification).
Polymorphism: (synonym: polymorphis allele) Natural variations in a gene, DNA sequence, or chromosome that have no adverse effects on the
individual and occur with fairly high frequency in the general population.
Polyploidy: An increase in the number of haploid sets (23) of chromosomes in a cell. Triploidy refers to three whole sets of chromosomes in a
single cell (in humans, a total of 69 chromosomes per cell); tetraploidy refers to four whole sets of chromosomes in a single cell (in humans, a
total of 92 chromosomes per cell).
Population risk: (synonym: background risk) The proportion of individuals in the general population who are affected with a particular disorder
or who carry a certain gene; often discussed in the genetic counseling process as a comparison to the patient's personal risk given his or her
family history or other circumstances.
Positional cloning: (synonym: reverse genetics) The cloning or identification of a gene for a particular disease based on its location in the
genome, determined by a collection of methods including linkage analysis, genomic (physical) mapping, and bioinformatics, when no
information about the biochemical basis of the disease is known; distinguished from the more common strategy of gene cloning beginning with
a known protein product, determining its amino acid sequence, and using that information to isolate the gene.
Positive predictive value: (synonym: PPV) The likelihood that an individual with a positive test result actually has the particular gene in
question, is affected, or will develop the disease.
Post-zygotic event: A mutational event or abnormality in chromosome replication/segregation that occurs after fertilization of the ovum by the
sperm, often leading to mosaicism (two or more genetically distinct cell lines within the same organism).
Prader-Willi syndrome: A condition characterized by obesity and insatiable appetite, mental deficiency, small genitals, and short stature. May be
deletion of #15 chromosome.
Predisposition: To have a tendency or inclination towards something in advance.
Predisposing mutation: (synonym: susceptibility gene) A gene mutation that increases an individual's susceptibility or predisposition to a certain
disease or disorder. When such a mutation is inherited, development of symptoms is more likely but not certain.
Predispositional testing: Testing of an asymptomatic individual in whom the discovery of a gene mutation indicates that eventual development
of findings related to a specific diagnosis is likely but not certain. A negative result may not exclude the possibility of future development of the
disease from other causes.
Preimplantation diagnosis: (synonym: preimplantation testing) A procedure used to decrease the chance of a particular genetic condition for
which the fetus is specifically at risk by testing one cell removed from early embryos conceived by in vitro fertilization and transferring to the
mother's uterus only those embryos determined not to have inherited the mutation in question.
Premutation: (synonym: intermediate allele) In disorders caused by trinucleotide repeat expansions, an abnormally large allele that is not
associated with clinical symptoms but that can expand into a full mutation when transmitted to offspring (Full mutations are associated with
clinical symptoms of the disorder).
Prenatal diagnosis: (synonym: prenatal testing) Testing performed during pregnancy to determine if a fetus is affected with a particular
disorder. Chorionic villus sampling (CVS), amniocentesis, periumbilical blood sampling (PUBS), ultrasound, and fetoscopy are examples of
procedures used either to obtain a sample for testing or to evaluate fetal anatomy.
Presymptomatic testing: Testing of an asymptomatic individual in whom the discovery of a gene mutation indicates certain development of
findings related to a specific diagnosis at some future point. A negative result excludes the diagnosis.
Presymptomatic diagnosis: Diagnosis of a genetic condition before the appearance of symptoms.
Primer: A short stretch of DNA (oligonucleotide) used in the polymerase chain reaction to initiate DNA synthesis at a particular location.
Private mutation: (synonym: unique mutation) A rare disease-causing mutation observed in a few families.
Probability: The long term frequency of an event relative to all alternative events, and usually expressed as decimal fraction.
Proband: (synonyms: index case, propositus) The affected individual through whom a family with a genetic disorder is ascertained; may or may
not be the consultand (the individual presenting for genetic counseling).
Probe: A specific, pre-fabricated sequence of DNA or RNA, labeled by one of several methods, used to detect the presence of a complimentary
sequence by binding (hybridizing) to it.
Prognosis: Prediction of the course and probable outcome of a disease.
Promoter region: A specific region just upstream from a gene that acts as a binding site for transcription factors and RNA polymerase during the
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initiation of transcription.
Protein analysis: One of several different testing methods that reveals either the structure or function of a particular protein product.
Protein functional assay: Measurement of the rate of a chemical reaction that takes place in the presence of an enzyme contained in a sample
taken from an individual. Reduced enzymatic activity may indicate carrier status or the diagnosis of a particular genetic disease
Protein truncation testing: (synonym: PTT) Means of identifying the shortened (truncated) proteins that result from mutations that specifically
cause premature termination of mRNA translation.
Proteus syndrome: A condition characterized by distorted asymmetric growth of the body and enlarged head, enlarged feet, multiple nevi on the
skin; mode of inheritance is unknown.
Predictive testing: Testing offered to asymptomatic individuals with a family history of a genetic disorder and a potential risk of eventually
developing the disorder.
Pseudogene: A copy of a gene that usually lacks introns and other essential DNA sequences necessary for function. Pseudogenes, though
genetically similar to the original functional gene, are not expressed and often contain numerous mutations.
Radiosensitivity testing: (synonyms: colony survival essay, CSA) Testing specific to ataxia-telangiectasia (A-T) and other disorders in which
cells are particularly sensitive to ionizing radiation; demonstrates colony formation of a blood lymphocyte cell line following irradiation, which is
abnormal in approximately 99% of patients with clinically diagnosed A-T, though the sensitivity of such testing in light of a suspected but
unsure diagnosis of A-T has not yet been documented.
Reading frame: (synonym: exon) A sequence of messenger RNA that is translated into an amino acid chain, three bases at a time, each triplet
sequence coding for a single amino acid.
Rearrangement: A structural alteration in a chromosome, usually involving breakage and reattachment of a segment of chromosome material,
resulting in an abnormal configuration; examples include inversion and translocation.
Recessive: A gene that is phenotypically manifest in the homozygous state but is masked in the presence of a dominant allele.
Reciprocal translocation: A segment of one chromosome is exchanged with a segment of another chromosome of a different pair.
Recombination: (synonym: crossing over) The exchange of a segment of DNA between two homologous chromosomes during meiosis leading to
a novel combination of genetic material in the offspring.
Recurrence risk: The likelihood that a trait or disorder present in one family member will occur again in other family members in the same or
subsequent generations.
Reflex testing: Follow-up testing automatically initiated when certain test results are observed in the laboratory; used to clarify or elaborate on
primary test results.
Repeat sequences: The length of a nucleotide sequence that is repeated in a tandem cluster.
Replication analysis: (synonyms: replication banding, X-chromosome inactivation study, XCI) A cytogenetic technique that uses specialized
banding procedures (replication banding) to identify the late-replicating (inactive) X chromosome in cells. Because it is less complicated, less
expensive, and less subjective, molecular testing is now used more commonly than replication analysis for X-chromosome inactivation studies.
Restriction fragment length polymorphism: (synonym: RFLP) Natural (polymorphic) variation in DNA sequence between an individual that
abolishes or creates endonuclease restriction (cutting) sites, resulting in DNA fragments of different lengths when DNA is digested by an
endonuclease.
Restriction fragment length polymorphism analysis: (synonyms: RFLP analysis, RFLP testing) Fragment of DNA of predictable size resulting from
digestion (cutting) of a strand of DNA by a given restriction enzyme. DNA sequence alterations (mutations) that destroy or create the sites at
which a restriction enzyme cuts DNA change the size (and number) of DNA fragments resulting from digestion by a given restriction enzyme.
Restriction site: A sequence of DNA that is recognized by an endonuclease (a protein that cuts DNA) as a site at which the DNA is to be cut.
Retinitis pigmentosa: Group of hereditary ocular disorders with progressive retinal degeneration. Autosomal dominant, autosomal recessive,
and x-linked forms.
Retinoblastoma: A childhood malignant cancer of the retina of the eye.
Reverse transcriptase: A viral enzyme used to make cDNA from mRNA.
RFLP: Restriction fragment length polymorphism; variations occurring within a species in the length of DNA fragments generated by a species
endonuclease.
Ribosomal protein: One of the ribonucleoprotein particles that are the sites of translation.
Risk assessment: Calculation of an individual's risk, employing appropriate mathematical equations, of having inherited a certain gene
mutation, of developing a particular disorder, or of having a child with a certain disorder based upon analysis of multiple factors including family
medical history and ethnic background.
RNA: (synonym: ribonucleic acid) The molecule synthesized from the DNA template; contains the sugar ribose instead of deoxyribose, which is
present in DNA; three types of RNA exist, messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA)
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Robertsonian translocation: The joining of two acrocentric chromosomes at the centromeres with loss of their short arms to form a single
abnormal chromosome; acrocentric chromosomes are the Y chromosome and chromosome numbers 13, 14, 15, 21, and 22
Rubinstein-Taybi syndrome: Condition with multiple congenital anomalies including: mental deficiency, broad thumbs, small head, broad nasal
bridge and beaked nose.
Sanger sequence: "Plus and minus" or "primed synthesis" method; DNA is synthesized so it is radioactively labeled and the reaction terminates
specifically at the position corresponding to a given base.
Second-degree relative: Any relative who is two meioses away from a particular individual in a pedigree; a relative with whom one quarter of an
individual's genes is shared (i.e., grandparent, grandchild, uncle, aunt, nephew, niece, half-sibling)
Segregation: The separation of the homologous chromosomes and their random distribution to the gametes at meiosis
Selection: The process of determining the relative share allotted individuals of different genotypes in the propagation of a population; the
selective effect of a gene can be defined by the probability that carriers of the gene will reproduce.
Sensitivity: The frequency with which a test yields a positive result when the individual being tested is actually affected and/or has the gene
mutation in question
Sequence alteration: (synonym: mutation) Any alteration in a gene from its natural state; may be benign (commonly referred to as a
"polymorphism"), pathogenic, or of unknown significance
Sequence analysis: (synonyms: gene sequencing, sequencing) Process by which the nucleotide sequence is determined for a segment of DNA
Sex determination: The mechanism in a given species by which sex is determined; in many species sex is determined at fertilization by the
nature of the sperm that fertilizes the egg.
Sickle cell anemia: An hereditary, chronic form of hemolytic anemia characterized by breakdown of the red blood cells; red blood cells undergo
a reversible alteration in shape when the oxygen tension of the plasma falls slightly and a sickle-like shape forms.
Simplex case: A single occurrence of a disorder in a family
Single-stranded conformational polymorphism: (synonym: SSCP) A type of mutation scanning; the identification of abnormally migrating singlestranded DNA segments on gel electrophoresis
Sister chromatid exchange: (synonym: SCE) Exchange of genetic material between the two chromatids of a single chromosome during the cell
division process; similar to crossing-over (recombination), except that the exchange involves the two sister chromatids of a single chromosome,
whereas crossing-over refers to exchange of genetic material between the two homologous chromosomes of a chromosome pair
Somatic cell hybrid: Hybrid cell line derived from two different species; contains a complete chromosomal complement of one species and a
partial chromosomal complement of the other; human/hamster hybrids grow and divide, losing human chromosomes with each generation until
they finally stabilize, the hybrid cell line established is then utilized to detect the presence of genes on the remaining human chromosome.
Somatic mutation: A mutation occurring in any cell that is not destined to become a germ cell; if the mutant cell continues to divide, the
individual will come to contain a patch of tissue of genotype different from the cells of the rest of the body.
Southern blotting: A technique for transferring electrophoretically resolved DNA segments from an agarose gel to a nitrocellulose filter paper
sheet via capillary action; the DNA segment of interest is probed with a radioactive, complementary nucleic acid, and its position is determined
by autoradiography.
Specificity: The frequency with which a test yields a negative result when the individual being tested is actually unaffected and/or does not have
the gene mutation in question
Spina bifida: A congenital condition that results from altered fetal development of the spinal cord, part of the neural plate fails to join together
and bone and muscle are unable to grow over this open section.
Splice-site mutation: A mutation that alters or abolishes the specific sequence denoting the site at which the splicing of an intron takes place.
Such mutations result in one or more introns remaining in the mature messenger RNA and can disrupt the generation of the protein product
Splicing: (synonym: splicing mutation) The process by which introns, non-coding regions, are excised out of the primary messenger RNA
transcript and exons (i.e., coding regions) are joined together to generate mature messenger RNA
Sporadic: The chance occurrence of a disorder or abnormality that is not likely to recur in a family
SSCP: (synonym: single-stranded conformational polymorphism) A type of mutation scanning; the identification of abnormally migrating singlestranded DNA segments on gel electrophoresis
Subtelomeric FISH screen: Uses DNA probes that are specific for the subtelomeric areas on the long arm and short arm of each chromosome,
allowing for the detection of cryptic and submicroscopic subtelomeric deletions and translocations, a significant cause of moderate to severe
mental retardation
Subtelomeric region: The chromosomal region just proximal to the telomere (end of the chromosome) composed of highly polymorphic
repetitive DNA sequences that are typically situated adjacent to gene-rich areas. Microdeletions and subtle rearrangements that disrupt genes
in the subtelomeric regions can cause mental retardation; use of fluorescent in situ hybridization (FISH) to evaluate subtelomeric regions is
usually required for detection of these abnormalities.
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Supernumary chromosome: A small chromosome containing a centromere occasionally seen in tissue culture, often in a mosaic state (present
in some cells but not in others). A marker chromosome may be of little clinical significance or, if it contains material from one or both arms of
another chromosome, may create an imbalance for whatever genes are present; assessment to establish the clinical significance, particularly if
found in a fetal karyotype, is often difficult.
Susceptibility gene: A gene mutation that increases the likelihood that an individual will develop a certain disease or disorder. When such a
mutation is inherited, development of symptoms is more likely but not certain
Syndrome: A recognizable pattern or group of multiple signs, symptoms or malformations that characterize a particular condition; syndromes
are thought to arise from a common origin and result from more than one developmental error during fetal growth.
Tay-Sachs disease: A fatal degenerative disease of the nervous system due to a deficiency of hexosamidase A, causing mental deficiency,
paralysis, mental deterioration, and blindness; found primarily but not exclusively among Ashkenazi Jews. Autosomal recessive.
Telomere: The segment at the end of each chromosome arm that consists of a series of repeated DNA sequences that regulate chromosomal
replication at each cell division. Some of the telomere is lost each time a cell divides, and eventually, when the telomere is gone, the cell dies.
Teratogens: Any agent that raises the incidence of congenital malformations.
3' – end: The end of a polynucleotide with a free (or phosphorylated) 3' - hydroxyl group.
Trait: Any detectable phenotypic property of an organism.
Trans configuration: (synonym: repulsion) Term that indicates that an individual who is heterozygous at two neighboring loci has the two
mutations in question on each of the two homologous chromosomes
Transcription: The formation of an RNA molecule upon a DNA template by complementary base pairing.
Transcription factor: (synonym: zinc finger protein) A protein that aids in the activation and regulation of transcription, in which messenger RNA
is synthesized from the DNA template; zinc finger proteins are one type of transcription factor.
Transduction: The transfer of bacterial genetic material from one bacterium to another using a phage as a vector.
Transferase: Enzymes that catalyze the transfer of functional groups between donor and acceptor molecules.
Transgenic organism: One into which a cloned genetic material has been experimentally transferred, a subset of these foreign gene express
themselves in their offspring. Turner syndrome a chromosomal condition in females (usually 45,XO) due to monosomy of the X- chromosome;
characterized by short stature, failure to develop secondary sex characteristics, and infertility.
Translation: The formation of a polypeptide chain in the specific amino acid sequence directed by the genetic information carried by mRNA.
Translocation: (synonym: chromosome rearrangement) A chromosome alteration in which a whole chromosome or segment of a chromosome
becomes attached to or interchanged with another whole chromosome or segment, the resulting hybrid segregating together at meiosis;
balanced translocations (in which there is no net loss or gain of chromosome material) are usually not associated with phenotypic abnormalities,
although gene disruptions at the breakpoints of the translocation can, in some cases, cause adverse effects, including some known genetic
disorders; unbalanced translocations (in which there is loss or gain of chromosome material) nearly always yield an abnormal phenotype
Trinucleotide repeat: Sequences of three nucleotides repeated in tandem on the same chromosome a number of times. A normal, polymorphic
variation in repeat number with no clinical significance commonly occurs between individuals; however, repeat numbers over a certain threshold
can, in some cases, lead to adverse effects on the function of the gene, resulting in genetic disease.
Triplet code: A code in which a given amino acid is specified by a set of three nucleotides.
Trisomy: The presence of a single extra chromosome, yielding a total of three chromosomes of that particular type instead of a pair. Partial
trisomy refers to the presence of an extra copy of a segment of a chromosome.
Trisomy rescue: The phenomenon in which a fertilized ovum initially contains 47 chromosomes (i.e., is trisomic), but loses one of the trisomic
chromosomes in the process of cell division such that the resulting daughter cells and their descendants contain 46 chromosomes, the normal
number
Tumor suppressor gene: Genes that normally function to restrain the growth of tumors; the best understood case is for hereditary
retinoblastoma.
Unaffected: An individual who does not manifest any symptoms of a particular condition
Unequal crossing over: Mispairing and exchange of DNA between genetically similar, nonhomologous chromosome regions that results in
duplication or deletion of DNA in each daughter cell
Uniparental disomy: (synonym: UPD) The situation in which both members of a chromosome pair or segments of a chromosome pair are
inherited from one parent and neither is inherited from the other parent; uniparental disomy can result in an abnormal phenotype in some
cases
Uniparental disomy study: (synonyms: UPD analysis, UPD study) Testing used to identify if specific chromosomes or chromosomal segments are
maternally or paternally derived; can aid in confirming the clinical diagnosis of certain disorders for which UPD is a possible underlying etiology
Variable expressivity: Variation in clinical features (type and severity) of a genetic disorder between affected individuals, even within the same
family
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Variable number tandem repeats: (synonym: VNTR) Linear arrangement of multiple copies of short repeated DNA sequences that vary in length
and are highly polymorphic, making them useful as markers in linkage analysis
VNTR: Variable number tandem repeats; any gene whose alleles contain different numbers of tandemly repeated oligonucleotide sequences.
Vector: A self-replicating DNA molecule that transfers a DNA segment between host cells.
Von Hippel-Lindau syndrome: An autosomal dominant condition characterized by the anomalous growth and proliferation of blood vessels on
the retina of the eye and the cerebellum of the brain; cysts and cancers in the kidneys, pancreas, and adrenal glands.
Western blot: The separation of proteins on an electrophoretic gel for identification by immunological techniques
Western blotting analysis: A technique used to identify a specific protein; the probe is a radioactively labeled antibody raised against the protein
in question.
Wild-type allele: The normal, as opposed to the mutant, gene or allele
X-chromosome inactivation: (synonyms: lyonization, XCI) In females, the phenomenon by which one X chromosome (either maternally or
paternally derived) is randomly inactivated in early embryonic cells, with fixed inactivation in all descendant cells; first described by the
geneticist Mary Lyon
X-inactivation: The repression of one of the two X-chromosomes in the somatic cells of females as a method of dosage compensation; at an
early embryonic stage in the normal female, one of the two X-chromosomes undergoes inactivation, apparently at random, from this point on
all descendent cells will have the same X-chromosome inactivated as the cell from which they arose, thus a female is a mosaic composed of two
types of cells, one which expresses only the paternal X-chromosome, and another which expresses only the maternal X-chromosome.
X-chromosome inactivation study: (synonym: XCI study) Molecular genetic testing to assess the relative proportion of methylated (inactive) X
chromosomes to unmethylated (active) X chromosomes; used to determine if X-chromosome inactivation is random or skewed
X-linked dominant: Describes a dominant trait or disorder caused by a mutation in a gene on the X chromosome. The phenotype is expressed in
heterozygous females as well as in hemizygous males (having only one X chromosome); affected males tend to have a more severe phenotype
than affected females.
X-linked lethal: A disorder caused by a dominant mutation in a gene on the X chromosome that is observed almost exclusively in females
because it is almost always lethal in males who inherit the gene mutation
X-linked recessive: A mode of inheritance in which a mutation in a gene on the X chromosome causes the phenotype to be expressed in males
who are hemizygous for the gene mutation (i.e., they have only one X chromosome) and in females who are homozygous for the gene mutation
(i.e., they have a copy of the gene mutation on each of their two X chromosomes). Carrier females who have only one copy of the mutation do
not usually express the phenotype, although differences in X-chromosome inactivation can lead to varying degrees of clinical expression in
carrier females
XYY syndrome: Genetic condition in males with extra Y chromosome (in 1 in 1000 male births). Symptoms: tall stature (over 6'), may including
sterility, developmental delay, learning problems.
YAC: Yeast artificial chromosome; a linear vector into which a large fragment of DNA can be inserted; the development of YAC's in 1987 has
increased the number of nucleotides that can be cloned.
Zygosity testing: The process through which DNA sequences are compared to assess whether individuals born from a multiple gestation (twins,
triplets, etc.) are monozygotic (identical) or dizygotic (fraternal); often used to identify a suitable donor for organ transplantation or to estimate
disease susceptibility risk if one sibling is affected
Zoo blot: Northern analysis of mRNA from different organisms.
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