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Detection method of nucleotide mutation points of KRAS gene and/or BRAF gene

A mutation site and detection method technology, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of low efficiency and high cost of detection methods, and improve accuracy and detection efficiency , save time and cost, and simplify operating procedures

Active Publication Date: 2010-09-22
BGI GENOMICS CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] The object of the present invention is to provide a detection method for the nucleotide mutation site of KRAS gene and / or BRAF gene, aiming to solve the problem of low efficiency and high cost of existing detection methods

Method used

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  • Detection method of nucleotide mutation points of KRAS gene and/or BRAF gene
  • Detection method of nucleotide mutation points of KRAS gene and/or BRAF gene
  • Detection method of nucleotide mutation points of KRAS gene and/or BRAF gene

Examples

Experimental program
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Embodiment 1

[0092] Embodiment 1: Detection of the nucleotide mutation site of the KRAS gene

[0093] (1) Design amplification primers for amplifying codons 12, 13, 61 and 146 of the KRAS gene and extension primers for detecting nucleotide mutations at each site

[0094] The wild type of codon 12 of KRAS gene is GGT, and its common mutation types include CGT, AGT, TGT and GCT, GAT, GTT; the wild type of KRAS gene codon 13 is GGC, and its common mutation types include CGC, AGC, TGC and GCC, GAC, GTC; the wild-type codon 61 of the KRAS gene is CAA, and its common mutation types include AAA, GAA, CCA, CGA, CTA, CAC, and CAT; the wild-type codon 146 of the KRAS gene is GCA, and its common mutations Types include ACA, CCA, GTA.

[0095] For the mass spectrometry detection of the above mutation sites, according to the KRAS gene sequence (NCBI accession number: EU332849), design each amplification primer and extension primer, each amplification primer has a tag sequence of 10 bases acgttggatg at...

Embodiment 2

[0113] Example 2: Simultaneous detection of each mutation site of the KRAS and BRAF genes.

[0114] In this embodiment, the 12th, 13th, 61st and 146th codons of the KRAS gene and the 600th codon of the BRAF gene were detected simultaneously.

[0115] (1) Design the amplification primer used to amplify the 600th codon of the BRAF gene and the extension primer used to detect the nucleotide mutation of this codon

[0116] The wild type of the 600th codon of the BRAE gene is GTG, and its common mutation types include GAG ​​and GCG. According to the BRAF gene sequence (NCBI accession number: NG_007873), each amplification primer and extension primer were designed, and each amplification primer had a tag sequence of 10 bases acgttggatg at the 5' end. The amplification primers used to amplify each codon of the KRAS gene are the same as in Example 1. The sequences and sequence numbers of the various amplification primers and extension primers are shown in Table 1 above.

[0117] (2...

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Abstract

The invention provides a method for detecting the nucleotide mutation points of a KRAS gene and / or a BRAF gene, which comprises the following steps that: (1) the mutation points to be detected of the KRAS gene and / or the BRAF gene are determined; (2) according to the mutation points, an amplification primer and the extension primer of each mutation point are designed; (3) PCR amplification; (4) SAP enzyme treatment; (5) extension reaction; (6) resin is adopted to purify an extension reaction product; and (7) mass spectrometry detection, the mutant of the target sites of the KRAS gene and / or the BRAF gene to be detected is determined. The method solves the problems of low sensitivity, limited accuracy, low flux and high cost of a traditional detection method. In addition, the invention also provides a specific primer and a primer combination of the method and the purposes thereof for the detection method.

Description

technical field [0001] The invention belongs to the field of organic biomolecules in genomics and molecular biology, and in particular relates to a detection method for nucleotide mutation sites of KRAS gene and / or BRAF gene. Background technique [0002] The genetic information of most organisms in nature is contained in deoxyribonucleic acid (DNA). There are 3 billion bases in the human genome, about 40,000 genes, distributed on 24 pairs of chromosomes. Each gene encodes a specific protein through transcription and translation, regulates a specific biochemical reaction in an organism, and exerts a specific biological function. Changes in the DNA sequence, that is, gene mutations, can lead to changes or loss of protein structure and function, which in turn can cause related genetic diseases. Gene mutations include insertions, deletions, and changes of base pairs in DNA molecules (point mutations). [0003] Studies have shown that the occurrence of many diseases, such as h...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N27/64C12N15/11
Inventor 韦清王威高扬李晶晶吴平王丽萍吕芳易鑫喻爽聂喜芳李国宏易吉朱江阳刘兴旺张俊青朱德琴张秀芝田超叶葭华桑杨玲
Owner BGI GENOMICS CO LTD
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