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New bidirectional one-step genotyping method through PCR

A genotyping and new method technology, applied in biochemical equipment and methods, microbial measurement/testing, etc., can solve problems such as high operating costs and complex equipment

Inactive Publication Date: 2011-01-19
SHENYANG BAICHUANGTE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the technology and equipment involved are very professional and complex, and the operating cost is very high

Method used

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  • New bidirectional one-step genotyping method through PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] A two-way one-step PCR genotyping method for the determination of homozygous A / A genotype DNA ( figure 1 T1 in

[0040] Proceed as follows:

[0041] a) According to the genotype A / A of the SNP site, design 5' end template mismatched specific PCR primers pA and pC;

[0042] b) Using specific PCR primers pA and pC that do not match the template at the 5' end, perform PCR from the SNP site in two directions at the same time;

[0043] c) Design two outer-end primers p1 and p2 at different lengths at the far end of the SNP site, whose 5'-3' points to the SNP site from the far end of the SNP site. Used to control the length of PCR products;

[0044] d) Qualitative or quantitative detection by electrophoresis and fluorescent probes.

[0045] The PCR reaction volume is 25 microliters. The pH value of the Tris-HCl buffer system is 8.3. The concentration of Mg ions is 2.5mM, the concentration of dNTP is 200uM, the outer primers are p1 and p2, and the specific primers are pA an...

Embodiment 2

[0048] A two-way one-step PCR genotyping method for the determination of homozygous G / G genotype DNA ( figure 1 T2 in

[0049] Step is with embodiment 1

[0050] The primers are still p1, p2, pA and pC. The PCR reaction volume is 25 microliters. The pH value of the buffer system containing Tris-HCl is 8.3. The concentration of Mg ions is 2.5mM, the concentration of dNTP is 200uM, the concentration of each primer is 200nM, and the KlenTaq DNA polymerase used in the reaction is 1.2u in the total reaction system, 2ng of DNA was used as a template. Negative control samples replaced the DNA template with water.

[0051] Electrophoresis results: only a 300bp PCR product synthesized by primers p1 and p2 and a 100bp PCR product synthesized by primers pC and p1. Negative control samples were water free of PCR products.

Embodiment 3

[0053] Two-way one-step PCR genotyping method to determine heterozygous A / G genotype DNA ( figure 1 T1 and T2 in

[0054] Step is with embodiment 1

[0055] The primers are still p1, p2, pA and pC. The PCR reaction volume is 25 microliters. The pH value of the buffer system containing Tris-HCl is 8.3. The concentration of Mg ions is 2.5mM, the concentration of dNTP is 200uM, the concentration of each primer is 200nM, and the KlenTaq DNA polymerase used in the reaction is 1.2u in the total reaction system, 2ng of DNA was used as a template. Negative control samples replaced the DNA template with water.

[0056] Electrophoresis results: There are PCR products synthesized by all primers. That is, a 300bp PCR product synthesized by primers p1 and p2, a 100bp PCR product synthesized by primers pC and p1, and a 200bp PCR product synthesized by primers pA and p2. Negative control samples were water free of PCR products.

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Abstract

The invention relates to a new bidirectional one-step genotyping method through PCR. The invention adopts the following technical scheme: a) designing a 5' terminal template mismatched or 5' terminal closed specific PCR primer according to the genotype of an SNP locus; b) adopting the 5' terminal template mismatched or 5' terminal closed specific PCR primer to simultaneously carry out PCR toward two directions respectively from the SNP locus; c) using an external terminal primer at the far terminal of the SNP locus to control the length of the PCR products; and d) detecting the PCR products in two directions according to the difference of the length of the PCR products in two directions and the difference of DNA sequences, thus obtaining the SNP result. The method designs the specific primer aiming at the genotype mainly related to the shape and can simultaneously determine the genotypes of a single or a plurality of loci by PCR. Each electrophoretic band represents the genotype of a locus. The results of the genotypes in different positions can be given by the integral electrophoregram.

Description

technical field [0001] The invention relates to the field of single-site or multi-site SNP assay methods, in particular to a method for detecting SNP by using a two-way one-step PCR genotyping method. Background technique [0002] SNP (single nucleotide polymophism), that is, single nucleotide polymorphism, is a nucleic acid sequence polymorphism caused by a single nucleotide change. Generally speaking, there are only two alleles at a SNP locus, so it is also called biallelic. SNP has a relatively high frequency of occurrence in the human genome, with an average of one polymorphic site in every 1000 bases. It is the third generation of genetic markers after microsatellites. Some SNP sites can also affect the function of genes, resulting in changes in biological traits and even disease. [0003] SNP research includes SNP discovery (discovery), SNP verification (validation) and SNP screening (Screening or Scoring). It is widely used in the study of population genetics (such...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 李伟东夏东元
Owner SHENYANG BAICHUANGTE BIOTECH
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