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Method for searching for new mutation/SNP locus based on double-nucleotide pyrosequeneing-by-synthesis

A two-nucleotide, pyrosequencing technology, applied in the biological field, can solve problems such as not easy to find, and achieve the effect of increasing the length of measurement and broadening the scope of analysis

Active Publication Date: 2016-01-20
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this way, the sequencing information of new mutations / SNPs is "irregularly" scattered in the sequencing information of high-abundance normal DNA templates, which is still not easy to be found

Method used

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  • Method for searching for new mutation/SNP locus based on double-nucleotide pyrosequeneing-by-synthesis
  • Method for searching for new mutation/SNP locus based on double-nucleotide pyrosequeneing-by-synthesis
  • Method for searching for new mutation/SNP locus based on double-nucleotide pyrosequeneing-by-synthesis

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Experimental program
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Embodiment 1

[0032] 1) Genomic DNA extraction: According to the source of the sample, select mature experimental steps to extract genomic DNA from standard samples and mixed samples.

[0033] 2) PCR: Design a pair of PCR primers, one of which is modified with biotin at 5', and amplify the fragment to be sequenced.

[0034] 3) Sequencing: The PCR amplification products of the standard sample and the mixed sample were divided into two parts, and reacted with streptavidin-modified magnetic beads to prepare a single-stranded DNA template; the sequencing primers were hybridized with the single-stranded DNA template, Specific two-nucleotide synthetic pyrosequencing was performed.

[0035] 4) Correlation analysis: Correct the pyrosequencing information of the two nucleotide synthesis, and perform division and subtraction operations on the two corrected mixed sample sequencing information and the known sequence samples to find out the differences in the information and derive The specific base ch...

Embodiment 2

[0054] A method for finding new mutations / SNPs from a sequence of the human sample UGT1A1 gene, the specific methods include:

[0055] (1) Select a single human blood sample confirmed by the sequence information as the standard sample, and 100 mixed blood samples from different people as the analysis sample;

[0056] (2) Genomic DNA in peripheral blood was extracted by traditional protein kinase K and phenol / chloroform extraction;

[0057] (3) PCR amplification: PCR primer 1: 5'-biotin-CCCTGCTACCTTTGTGGACT-3' (SEQ ID NO: 3), PCR primer 25'-CATTATGCCCGAGACTAACAAA-3' (SEQ ID NO: 4) polymerized with 200ng genomic DNA, 0.2mMdNTP, 1UTaqDNA Enzyme, 1× amplification buffer, 1.8mM MgCl 2 50 μL PCR amplification system was used for amplification. The amplification conditions were: initial denaturation at 95°C for 5 min; 40 thermal cycles: denaturation at 94°C for 30 s, annealing at 57°C for 45 s, extension at 72°C for 45 s, and final extension at 72°C for 7 min. Each sample was ampli...

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Abstract

The invention discloses a method for searching for a new mutation / SNP locus based on double-nucleotide pyrosequeneing-by-synthesis. According to the method, PCR is conducted on the genomic DNAs of a known sequence sample and a mixed sample, pyrosequeneing is conducted on obtained single-stranded PCR products according to a specific double-nucleotide adding method, each sample acquires two groups of double-nucleotide pyrosequeneing-by-synthesis information, and correction is conducted; the two groups of corrected mixed sample sequencing information are compared with the two groups of corrected known sequence sample sequencing information; if neither the two groups of mixed sample sequencing information nor the two groups of known sequence sample sequencing information are changed, it is indicated that the mixed sample sequencing information is consistent with the known sequence sample sequencing information, and no new mutation / SNP locus exists; if the mixed sample sequencing information is different from at least one of the two groups of known sequence sample sequencing information, it is indicated that the mixed sample sequencing information is not totally consistent with the known sequence sample sequencing information, and it shows that a new mutation / SNP locus exists.

Description

technical field [0001] The invention belongs to the field of biotechnology, and is a PCR product sequence analysis method, in particular to a method for discovering new mutations / SNP sites from mixed samples by performing synthetic pyrosequencing by adding specific nucleotides. Background technique [0002] The development and completion of the Human Genome Project and various model organism genome projects has brought mankind into the post-gene era, which has had a huge impact on contemporary biological and medical research, and the related disciplines of molecular biology have developed rapidly. It will become possible to understand the differences of life, the law of disease occurrence and development, and the interaction between drugs and living organisms at the genetic level. Among many gene variations, point mutation / SNP is an efficient quantitative marker when studying genetic inheritance and variation. Point mutations / SNPs are associated with both monogenic and poly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2561/101C12Q2563/149C12Q2563/143C12Q2565/301
Inventor 肖鹏峰殷豪景唐健
Owner SOUTHEAST UNIV
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