Kit, method and application for detecting mutation of predetermined locus in DNA sample

A technology for pre-determining sites and samples, which is applied in recombinant DNA technology, DNA/RNA fragments, biochemical equipment and methods, etc., and can solve problems such as specific site mutations that need to be improved.

Active Publication Date: 2012-04-11
BGI GENOMICS CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, in the field of molecular biology, the detection of mutations at specific sites still needs to be improved

Method used

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  • Kit, method and application for detecting mutation of predetermined locus in DNA sample
  • Kit, method and application for detecting mutation of predetermined locus in DNA sample
  • Kit, method and application for detecting mutation of predetermined locus in DNA sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] ①Primer design

[0107] According to the selected 20 deafness gene mutation sites to be detected and / or typed, including 4 genes, they are: GJB2 gene (including sites 35delG, 167delT, 176-191del16, 299_300delAT and 235delC), GJB3 gene ( Including sites 538C>T and 547G>A), SLC26A4 gene (including sites 281C>T, 589G>A, 919-2A>G, 1174A>T, 1226G>A, 1229C>T, 1975G>C, 2027T> A, 2162C>T, 2168A>G and IVS15+5G>A) and 1494C>T and 1555A>G of mt DNA.

[0108] Design an amplification primer pair and an extension primer according to the position and genotype of the mutation site, wherein the length of the amplification primer is about 30 bases, and the amplification primer has a tag sequence of 10 bases acgttggatg at the 5′ end; The length of the extension primer is 17-28 bases, allowing 1-5 bases at the 5' end to be incompletely paired with the template, and the bases at the 3' end are completely matched with the adjacent bases at the 3' end of the mutation site. In addition, the ...

Embodiment 2

[0139] ①Primer design

[0140]According to the selected 14 deafness gene mutation sites to be detected and / or typed, they are distributed in 3 genes, namely: GJB2 gene (including sites 299_300delAT and 235delC), SLC26A4 gene (including sites 281C>T , 919-2A>G, 1174A>T, 1226G>A, 1229C>T, 1975G>C, 2027T>A, 2162C>T, 2168A>G and IVS15+5G>A) and 1494C>T and 1555A of mt DNA >G. The amplification primers designed by the present invention for the above 14 mutation sites of deafness genes are selected from the primers in Example 1, see Table 7. The extension primers designed by the present invention for the above 14 mutation sites of deafness genes are selected from the primers in Example 1, see Table 8.

[0141] Table 7: Amplification primers for the above 14 mutation sites of deafness genes.

[0142]

[0143] Table 8: Extension primers for the above 14 mutation sites of deafness genes.

[0144]

[0145]

[0146] Subsequent experimental steps (namely: DNA extraction, PCR ...

Embodiment 3

[0153] ①Primer design

[0154] According to the selected 4 deafness gene mutation sites to be detected and / or typed, they are distributed in 3 genes, namely: GJB2 gene (including site 235delC), SLC26A4 gene (including site 919-2A>G ) and 1494C>T and 1555A>G of mt DNA. Refer to Table 9 for the amplification primers designed by the present invention for the above four mutation sites of deafness genes, and refer to Table 10 for the extension primers.

[0155] Table 9: Amplification primers for the above four mutation sites of deafness genes.

[0156]

[0157] Table 10: Extension primers for the above four mutation sites of deafness genes.

[0158] serial number

Primer sequence

Primer description

SEQ ID NO: 37

ggATCCTGCTATGGGCC

Extension primer for position 235delC

SEQ ID NO: 42

GCAGTAGCAATTATCGTC

Extension primer for position 919-2A>G

SEQ ID NO: 51

CGTACACACCGCCCGTCAC

Extension primer for position 1494C>T

S...

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Abstract

The invention relates to a kit, a method and an application for detecting mutation of predetermined locus in DNA sample. The method for detecting mutation of predetermined locus in DNA sample comprises the following steps: using an amplification primer for performing PCR amplification reaction on the DNA sample, using an extended primer and dd NTP; using the amplification product as template, performing the oligonucleotide extension reaction, so as to obtain the extended primer having a 3' end connected with a base, which is the extended product; and based on the molecular weight of the extended primer, determining the mutation type of the predetermined locus. The invention can effectively determine whether there is mutation in the predetermined locus and can determine the mutation type.

Description

technical field [0001] The invention relates to a kit, a primer combination, a method and an application for detecting a mutation at a predetermined site in a DNA sample. Background technique [0002] Forming mutations at specific sites is a commonly used research method in molecular biology experiments, which can help analyze the effect of point mutations at specific sites on gene function. Generally speaking, after constructing the target mutant, how to mutate the site correctly is a problem faced by those skilled in the art. [0003] However, in the field of molecular biology, the detection of mutations at specific sites still needs to be improved. Contents of the invention [0004] The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, an object of the present invention is to provide a method capable of effectively detecting mutations at predetermined sites in DNA samples. [0005] According to the first aspe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N27/62
CPCC12Q1/68C12Q1/6827C12Q2525/186C12Q2533/101C12Q2563/167
Inventor 冯大飞邹婧刘兴旺王夏曼汪建王俊杨焕明
Owner BGI GENOMICS CO LTD
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