Download Highlight of mutation GPS® technique

Mutation GPS® Gene Positioning System
Highlight of mutation GPS® technique
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Specificity: only amplify the specific targeted sequence(s)
Sensitivity: PCR based technique only need nanogram level DNA
Quantification: quantify the mutation percentage in one sample
Convenient: incubate overnight and run two PCRs to pick up the mutations
Fast: amplified the mutation sequences within one day
Introduction
A deletion is a mutation caused by loss of a DNA sequence. An insertion is a mutations caused by adding a piece of DNA into genome,
which can occur naturally, or can be artificially created for research purposes in the lab mediated by virus, plasmid or transposons.
Exogenous DNA insertion mutations and transposon jumping within genome have no a known favorable locations in host genome.
The mutagenesis caused by insertion or deletion is a fairly common occurrence and their effect severity on gene depends on both the
mutation sequence and the mutation location in genome. One type of the mutations may affect gene expression level. The other type of
mutation involved in reading frame can change the protein amino acid sequence and/or size, which will affect the function of the protein. The
altered protein expression level and/or sequence frequently are unable to function properly or can possibly result in diseases.
Currently already known many viruses insertion can cause many disease and cancers in human. DNA deletions also can cause many human
genetic disease, i.e. spinal muscular atrophy, von Willebrand disease, Cystic Fibrosis, Williams syndrome. Transposable Element jumping in
genome can cause many diseases including hemophilia A and B, severe combined immunodeficiency, porphyria, colon cancer, and
Duchenne muscular dystrophy.
Detection of insertion/deletion mutations generally is challenging, especially when the mutation location and size are unknown or varies
greatly. When mutations only occurred in some somatic cell i.e. cancers, finding these unknown mutations are the most important for
Precision medicine/Personalized medicine/Individualized medicine.
For the present, the methods used to detect these unknown insertion/deletion are either not sensitive or not convenient or not accurate or
expensive. Only run two PCR with this Mutation GPS, the insertion/deletion mutations can be specifically amplified from a mixed genomic
DNA sample, which overcomes the disadvantages mentioned above.
Principle and Technical outline
Msp-I is a restriction enzymes that recognize the DNA sequence
5’-CCGG. HarborgenaseTM (patent pending) is a novel ligase
that link adapter and DNA restriction fragment, but not
catalyses
the
ligations
between
fragments.
Using
HarborgenaseTM, Msp-I and a Y-shaped DNA adapter a fully
tagged restriction fragment library can be made, in which every
end of DNA restriction fragments will be linked an adaptor and
no fragment-fragment self ligation.
With the universal primer alone that match one arm of Y-shape
adapter, the amount of restriction fragments will be not amplified
in PCR (its’ number increased by arithmetic progression)
because of the unique sequence of the arms in Y-shaped
adapter. If using both the universal primer and a target
sequence specific primer, the target sequence will be amplified
in PCR ((it’s number increased by geometric progression and
non-target fragment by arithmetic progression). Generally, the
fragment ration between target fragment and non-target
fragment is about one over millions. This ratio will be reach to
about 1 about 30 cycles PCR later because the amplified way
differently between target fragment and non-target fragment.
Application
· Virus Insertion mutation
· Transposon jumping in genome
· Deletion mutation
· Gene Duplication
· ChromosomeTranslocation
Harborgen Biotechnology Company
4539 Metropolitan Court, Frederick, MD 21704 Phone: (240) 595-2652 Fax: (240) 751-9488
Email: [email protected]
Kit content and Price
kit size
25X
100X
Cat. #
GP101025
GP101100
Enzyme mix
15 ul
60 ul
5x Enzyme buffer
100 ul
400 ul
Universal Primer
30 ul
80 ul
2x Taq polymerase
750 ul
3,000 ul
Equipment and Reagents to Be Supplied by User
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Target specific primer, Samples, PCR instrument, tubes/plates, tips and PCR grade H2O
DNA purification system for PCR, agarose gel or polyacrylamide gel
NGS or Sanger sequencing system
Mutation GPS Protocols:
1, Protocol to make tagged DNA library (i.e. one gDNA sample)
Make following reaction into a 100 ul or 200 ul PCR tube. Gently mix it thoroughly with blending pipette tip.
purified DNA
10 ng to 200 ng
5X GPS buffer
4 ul
enzyme-Mix
0.6 ul
H2O
to 20 ul
Incubate the PCR tube in PCR instrument with following thermal protocol. Turn off the heat lid of the PCR instrument
(important!).
a. 25 degree for 2 min,
b. 4 degree for 2 min,
c. 16 degree for 1 min, 100 cycles from step a to step c,
d. 37 degree for 2 hours,
e. 4-degree holding.
2, Purify the library with commercial PCR purification kit
3, 1st PCR Protocol to enrich the target sequences
PCR reaction setting
Purified library
1-10 ng
100x Universal primer
0.2 ul
25uM target specific primer
0.2 ul
2x PCR mix
10 ul
H2O
to 20 ul
PCR thermal protocol
1. 95 °C 10 min
2. 95 °C 5 sec
3. 60-70 °C 60 sec, 30 cycles from step 2 to step 3
4. 72 °C 5 min
5. 4 °C holding
4, 2nd PCR Protocol to amplify the target sequences. Dilute the 1st PCR amplicon 10,000 times with H2O
10,000X Diluted 1st PCR amplicon
1.0 ul
100x Universal primer
0.2 ul
25uM target specific primer
0.2 ul
2x PCR mix
10 ul
H2O
8.6 ul
PCR thermal protocol:
1.
95 °C 10 min
2.
95 °C 15 sec
3.
60-70 °C 60 sec, 40 cycles from step 2 to step 3
4.
72 °C 5 min
5.
4 °C holding
5, Purify the 2nd PCR amplicon with the PCR, or agarose gel, or polyacrylamide gel purification kit.
6, Sequencing of the purified amplicon(s):
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Next Generation Sequencing with the universal primer for PCR purification samples
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Sanger sequencing with target specific primer or the universal primer for gel purified sample
Harborgen Biotechnology Company
4539 Metropolitan Court, Frederick, MD 21704 Phone: (240) 595-2652 Fax: (240) 751-9488
Email: [email protected]