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Method for polymorphism detection of gene based on single base extension reaction

A technology of single base extension and gene polymorphism, which is applied in the field of gene polymorphism detection based on single base extension reaction, can solve the problems of impact, increase of detection cost, fluorescent label detection results affected by fluorescence properties, etc., and achieve operational Simple, low cost, rapid detection effect

Inactive Publication Date: 2012-01-25
SUZHOU UNIV
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  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to provide a method for detecting gene polymorphism based on single-base extension reaction, which solves the problem that ddNTPs are required for labeling in the prior art when single-base extension reaction is detected, resulting in increased detection cost and fluorescent label detection results being affected by fluorescence. nature of the impact

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  • Method for polymorphism detection of gene based on single base extension reaction
  • Method for polymorphism detection of gene based on single base extension reaction
  • Method for polymorphism detection of gene based on single base extension reaction

Examples

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Effect test

Embodiment 1

[0038] Example 1 Determination of 100C>T site in CYP2D6 gene by high performance liquid chromatography

[0039] Isoquine 4-hydroxylase CYP2D6 is one of the members of the cytochrome P450 enzyme system, involved in the metabolism of more than 80 drugs, such as alprenolol, propranolol and so on. CYP2D6 gene polymorphisms have obvious effects on the metabolic activity of this enzyme. In this embodiment, the practicability of the present invention is studied by taking the SNP site 100C>T (rs1065852) as an example. In order to improve the detection sensitivity and reduce the amount of genomic DNA used, a pair of primers were first designed to amplify the DNA fragment containing the 100C>T site. The primer sequences were P1: 5′-AACGCT GGG CTG CAC GCT AC-3′ and P2: 5 '-TGG TCG AAG CAG TAT GGTGT-3', the length of the DNA fragment generated by amplification is 100bp; the 3'-end of primer P1 is close to the 100C>T site, so it is also used as a single base extension primer. Then go thr...

Embodiment 2

[0045] Example 2 Determination of 100C>T site in CYP2D6 gene by high performance liquid chromatography

[0046]This embodiment takes the insertion / deletion site 4657-4659ACA>del(rs78340630) in the CYP2D6 gene as an example to study the practicability of the present invention. In order to improve the detection sensitivity and reduce the amount of genomic DNA used, a pair of primers were first designed to amplify the DNA fragment containing the rs78340630 site, and the primer sequences were P3: 5′-TCC TGC CAG CAC CAT CAC A-3′ and P4: 5′ -AGGGAA CGT TCT GGC ACC T-3', the length of the DNA fragment generated by the amplification is 139bp; the 3'-end of the primer P3 is close to the rs78340630 site, so it is also used as a single base extension primer. Then go through a purification step to remove other components in the reaction solution except the target DNA fragment. Then, using the purified DNA fragment as a template, two deoxyribonucleotides dATP and dGTP corresponding to the...

Embodiment 3

[0051] Embodiment 3 high performance liquid chromatography measures allele frequency

[0052] Research and discovery of SNP sites related to the occurrence and development of diseases, or SNP sites related to differences in drug metabolism are often obtained by comparing the allele frequencies of the SNPs studied in two groups of people (disease population and normal population). Tens of thousands of samples often need to be analyzed, and the workload is very huge. Therefore, some high-throughput and low-cost SNP typing technologies are urgently needed to meet the demand. SNP analysis is still very expensive and time-consuming for large numbers of individuals. Therefore, an effective method to reduce testing costs and operations is to measure equal amounts of mixed DNA samples. The method of measuring equal pooled DNA samples reduces genotyping operations, and the ratio of wild-type / mutant alleles in DNA pooled samples can be accurately determined over a wide range. In this ...

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Abstract

The invention discloses a method for polymorphism detection of a gene based on single base extension reaction. The method is characterized by comprising the following steps: (1) designing and selecting a specific primer according to a gene sequence to be detected, adding the specific primer in a mixed liquid containing a sample to be detected, a dideoxynucleotide (ddNTP) pair or deoxynucleotide triphophate (dNTP) pair for single base extension reaction so that a base in ddNTP or dNTP is added at the terminal end of the specific primer, wherein the specific primer is positioned at the upstream of gene SPN (single nucleotide polymorphism) site to be detected, the 3'-terminal base of the primer is close to the SNP site to be detected, and a continuous nucleotide sequence is composed of 15-45 continuous nucleotides containing the gene sequence to be detected; and (2) detecting the amount of surplus ribonucleotides in an extended product, judging the type of the polymorphism of the target gene according to the amount of the ddNTP or dNTP in the extended product. The method has low cost and is simple to operate, and can rapidly detect the polymorphism of the gene in no need of complicated optimization steps.

Description

technical field [0001] The invention belongs to the technical field of gene polymorphism detection, and in particular relates to a method for detecting gene polymorphism based on a single base extension reaction. Background technique [0002] After the completion of the International Human Genome Project (HGP), scientists discovered in the process of deciphering the human genome that the genome sequences of any two different individuals have about 0.1% difference. Such gene sequence differences are called polymorphisms, the most prevalent of which are single nucleotide differences, namely single nucleotide polymorphisms (SNPs), which account for more than 90% of all known polymorphisms [Lander ES , Linton LM, Birren B, et al. Initial sequencing and analysis of the human genome. Nature 2001, 409(6822): 860-921.]. Gene polymorphisms determine differences in different races and groups, as well as differences in disease susceptibility and drug treatment sensitivity in different...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 汪维鹏
Owner SUZHOU UNIV
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