Download Mutational Profiling of Human Disease Genes

Mutational Profiling of Human Disease Genes
Soumya N idtha 1 , Richard R. Bennett2 Karen Poulter1, Rixun Fang 1, Robert N utter1, and Primo Baybayan1 , Applied Biosystems, Foster City, CA 944041 ; Division of Genetics, Children's H ospital, Boston, MA 2
ABSTRACT
Identification of mutations in human genes to determine the genetic basis of diseases is a challenge. A
combination of methods such as sequencing, Denaturing High Performance Liquid Chromatography
(DHPLC) and Single Strand Conformational Polymorphism (SSCP), are used to study mutation
profiles. However, even though sequencing is the most accurate and complete methodology for
mutation detection it is perceived to be difficult and expensive. To eliminate the time consuming step
of designing, optimizing and validating of PCR primers for resequencing human disease genes,
Applied Biosystems has developed the V ariantSEQr™ Resequencing System. This system can be
easily integrated into any sequencing pipeline. VariantSEQr™ takes advantage of the automated
capillary electrophoresis platform, reagents and SeqScape® v2.1.1 software for mutation detection and
report generation.
This fully integrated and optimized system allows researchers to study:
1. Disease genes with dense mutation spectrum and polygenic diseases
2. Larger cohorts and therefore conclusions drawn may be more statistically significant
We describe a new tool for mutation and SNP discovery that is easy to integrate into any sequencing
laboratory and takes advantage of the high degree of data accuracy provided by DNA sequencing.
INTRODUCTION
The wealth of data generated during the human genome project is very valuable to elucidate the
relationship between sequence variation and susceptibility to disease. M arkers such as SNPs (Single
Nucleotide Polymorphisms), insertions and deletions in the human genome are useful in two ways.
Firstly, the polymorphism can cause differences in the gene function or regulation that directly
contribute to disease processes or serve as markers because of associations with causative mutations.
However, it is still necessary to identify the type and location of the mutation in order to develop
strategies for treatment. Mutations important in many diseases are found in all regions of the gene,
including regulatory regions, exons and introns.
A number of methods to screen for putative point mutations, insertions and deletions exist today.
Some of the methods include DHPLC, SSCP, a variation of SSCP called DOVAM (Detection of
Virtually All Mutations), HA (Heteroduplex Analysis) and, DGGE (Denaturing Gradient Gel
Electrophoresis). Each technique assumes mutations are rare and therefore it is more economical to
screen for regions where mutations occur. However, each have significant shortcomings including
extensive primer design and PCR validation, allele-specific amplification due to SNPs in primer sites,
inconsistent data generation and no automated data analysis. Additionally, not all mutations are
reliably detected and recent publications show mutations occur, on average, once every 250-300
nucleotides in a population. This means in order to know the nature of putative mutations found by
screening methods, it is necessary to design and validate new primers and sequence each region
followed by very laborious analysis of the data.
Figure 1. Identification of mutations of
interest ultimately lead to Sequencing
RESULTS
In order to precisely determine the location and nature of mutations, Applied Biosystems developed the
VariantSEQr™ Resequencing System which employs DNA sequencing for simultaneous discovery and
genotyping of all mutations. VariantSEQr™ Resequencing System is a fully integrated system capable
of quickly resequencing human genes and Mitochondrial DNA in a cost effective manner. This system
consists of PCR primers of known performance. It can be fully integrated -detection of mutation
variation via sequencing through data analysis using all instruments, reagents and data analysis
software.
DHPLC is one of the methods used to screen samples for mutations. Upon mixing, denaturing
and reannealing of amplicons containing one or more mismatches characteristic peak patterns
for homozygous and heterozygous samples are obtained. For example in a study on a large
human gene, size~2.4million base pairs with mutations spanning all exons using 87 primers,
screening known mutations first by DHPLC, followed by sequencing for a more complete
mutation profile may not be most cost effective approach.
Figure 3. Reliable amplification and sequence data
using Universal PCR and Sequencing protocol
PCR amplicons generated using optimized PCR
primers that are tailed with M13 priming sites
and universal protocol for easy experimental set
up for projects of any size.
Figure 2. Workflow for each primer set when using DHPLC for
screening m utations
Sequence data using universal sequencing primers
and protocol.
Heterozygote variants are detected and reported
accurately by SeqScape® softwareV2.1.1.
MATERIALS AND METHODS
DHPLC:
Genom ic DNA Preparation- Assessed Genomic DNA Quality.
Ready -to-use Resequencing Sets for:
Large set of Kinase gene fam ily -cancer studies, Muscular dy strophy ,
Drug metabolism Enzy mes, A utoimmune, Inflammatory,
Cardiovascular, Metabolic Disorders, Mitochondrial DNA
STEP 1:PCR A mplification and WAVE® sy stem for DHPLC analysis
PCR primers were optimized for gene. PCR reactions were run on the validated PCR primers.
Unpurified PCR amplicons from specimens were mixed in a 1:1 ratio with an aliquot of unpurified PCR
amplicon from an unaffected specimen and run on WAVE system.
STEP 2:Sequence analy sis
PCR amplicons were purified using the QIAquick PCR Purification Kit (Qiagen, V alencia, Ca.).
DNA concentration was determined by spectrophotometry.
Unincorporated dye and other contaminants were removed with SEQueaky Kleen 96well kit
(BI O-RAD Laboratories, Hercules, Ca.) Both strands were sequenced on an ABI PRISM® 310 DNA
Sequencer
Data was analyzed with the SequencherTM (software (Genecodes Inc. Ann Arbor, Michigan).
VariantSEQrTM
Resequencing System:
Genom ic DNA Preparation- Assessed Genomic DNA Quality
STEP 1: PCR A mplification
PCR Reactions were prepared with validated resequencing primer pair for gene of interest. PCR
Reactions were run on the ABI GeneAmp® PCR System 9700. PCR products were cleaned up
using Exo-SAPI T
DNA Sequencing
Amount of PCR product was estimated on agarose gel
Sequencing Reactions were run. Extension products were purified and run on Applied
Biosystems 3730 DNA Analyzer
Data was analyzed using SeqScape® software V2.1.1 (Applied Biosystems, Foster City, CA)
Extensive Optimization required: for each prim er set
Optimizing PCR primers~1 week
Optimizing conditions for amplicon hybridization~1 week
Analyze DHPLC data~1week
data incomplete, optimize primers for sequencing~1 week
Analyze sequence data~1 week
Figure 3. Workflow using VariantSEQr™
Resequencing System
for a gene
Order on myScienceSM Research
Order on myScienceSM Research
Environment-web based gene catalog:
Environment-web based gene catalog:
Optimized and ready to use primer pairs for
Optimized and ready to use primer pairs for
Resequencing hum an genes
Resequencing hum an genes
PCR
PCR
Sequencing
Sequencing
VariantSEQr TM employs sequencing, a well
established and robust technique to screen
and discover genetic variation in 1,000s of
human genes. There are 3 sets offered for
each gene or Resequencing Se t (RSS)Complete
Coding
Non-coding
Universal PCR protocol
for all genes/RSS.
Universal sequencing prim ers and
protocol for all genes/RSS.
CONCLUSIONS
Sequencing is the most direct and complete method for the detection of all mutation making it an
efficient tool for routine resequencing projects of human genetic diseases for all types of mutations.
Methods that have been developed to first screen for mutations require considerable time to design and
optimize primers, optimize running conditions and analyze the data. Once putative mutations have
been identified, it is still necessary to sequence every region to identify all mutations This takes time
and money.
VariantSEQr™ Resequencing System uses sequencing:
•
Verification of well known mutations and discovery of rare mutations at the same time.
•
No primer optimization is necessary
•
All primers use a universal protocol
•
Detection of genetic changes as well as interpretation of results is rapid and accurate.
.
REFERENCES
1. Detection of muta tions in t he dys troph in g en e v ia auto mate d DH PL C screen ing an d direc t seq uencing
Rich ard R Bennett1 , Jo han den Dun nen2 , K ristine F O'Brien3 , Basi l T Darras4 and Louis M Kunk el1, 3, 5
BM C Gene tics 2001, 2 :17
1Division o f Genetics, Children 's Hospital, Boston , Massa chu setts, USA
2Center for Huma n an d Clinical Genetics, Leid en University Medi cal Cente r, Leiden , Ned erland
3Depa rtment of Ge netics, Har vard M edical S choo l, Boston, Massa chusetts, US A
4Depa rtment of Neuro logy, Chi ldren's Hosp ital, B oston, Massachusetts, USA
5How ard Hughes Medical Insti tute, Children 's Hospital, Ha rvard M edical Scho ol, Boston , Massachu setts, USA
2. Seq uence -Bas ed Link age A nalysis
Itay Furman,1 M ark J. Rie der,2 Suz ann e da Ponte,2 Dana P. Carrington,2
Deborah A. Nickerson,2 L eonid Kruglyak ,1,3 and Kyria cos Mark iano s1
1Divisio n of Human Biology, Fred Hutch inso n Cancer Research Ce nter, a nd 2 Dep artment of Ge nome Sciences, Un iver sity of Washi ngto n, Seattle; a nd 3 How ard Hughes
Medical Institute , Che vy Chase, MD
3. Meth od s in Mo lecular Biology : Sing le N ucleo tide P olymo rphisms, me tho ds an d prot oco ls. Vol 212 Edited b y Pui-Yan Kw ok
ACKNOWLEDGEMENTS
Data A naly sis
Data A naly sis
Powerful software for accurate
mutation call and reporting.
Thank s to Richa rd R B ennett for sharing his da ta and exp ertise on DHPLC. We w ou ld al so lik e to thank the Applie d Bi osy stems R&D tea m for help ing generate sequ ence
data.
TRADEMARKS/LICENSING
For Research Use On ly. Not for use in d iagnosti c procedures.
The PCR p roce ss is covered by patents ow ned by Roche M olecular Systems, In c. and F. Ho ffmann -La Roche, Ltd.
Appl ied B iosystems, ABI PRISM , and SeqScape are registe red trademark s and AB De sign , myScience, and VariantSEQr are trad ema rks of A pplera Corporation or its
subsidiaries i n the U.S. and/or certain oth er countries.
TaqM an a nd Ge neAmp a re reg istered tra demarks o f Roche Mo lecular Sys tems, Inc. A ll other trad ema rks a re the property of their r espe ctive ow ners.