Method and kit for detecting KRAS gene mutations in human colon and rectum cancers

A colorectal cancer, human detection technology, applied in the field of gene mutation detection, can solve problems such as indistinguishable

Inactive Publication Date: 2011-07-06
苏州科贝生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing technology still has not solved the fatal defect that HRM detection cannot be used for genotyping. Although up to 7 kinds of hotspot mutations can be detected at the same time, the specific sites of these mutations cannot be distinguished. In scientific research and clinical work, it is often necessary to Subsequent PCR enrichment and sequencing are used to genotype the detected mutations, and effectively distinguish the codon 12 and 13 mutations that are closely related to the prognosis of colorectal cancer and the efficacy of targeted therapy from other mutations

Method used

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  • Method and kit for detecting KRAS gene mutations in human colon and rectum cancers
  • Method and kit for detecting KRAS gene mutations in human colon and rectum cancers
  • Method and kit for detecting KRAS gene mutations in human colon and rectum cancers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Selection of samples and extraction of genomic DNA

[0060] The collected genomic DNA comes from whole blood, cells, fresh tissue, and paraffin-fixed tissue. The extraction method refers to the operation manual provided by the kit, and some optimizations have been made at the same time.

[0061] Whole blood genomic DNA of healthy people was used as the wild-type control of the experiment, and was extracted using the BloodGen Mini Kit of Kangwei Century;

[0062] Genomic DNA derived from the A549 cell line was used as a homozygous mutant (G34A) control for the experiment,

[0063] The genomic DNA derived from the HCT116 cell line was used as the heterozygous mutant (G38A) control of the experiment, and was extracted using the DNA FlexGen DNA Kit of Kangwei Century;

[0064]Both fresh tissue and paraffin tissue were derived from mice, used to evaluate the adaptability of the HRM method to fixed tissue, and extracted using DNA TissueGen DNA Kit of Kangwei Centu...

Embodiment 2

[0069] Example 2: Identification of mutation sites

[0070] The samples with known KRAS 12 and 13 codon genotypes were detected by HRM technology.

[0071] . Specific primers and probes are as follows:

[0072] SEQ ID No: 1, forward primer 1: 5'-GCCTGCTGAAAATGACTGAA-3'

[0073] SEQ ID No: 2, reverse primer 1: 5'-TATCGTCAAGGCACTCTTGC-3'

[0074] SEQ ID No: 7, specific probe: 5'-CTCTTGCCTACGCCACCAGCTCCAACT-3'.

[0075] . Amplify the fragments near codon 12 / 13 by PCR; prepare the mixture: add 1 μl of the previously prepared genomic DNA solution, 2 μl PCR buffer (10×), 1.6 μl dNTP, 0.1 μl HotStarTaq DNA polymerase, forward primer 0.4 μl and 0.08 μl of reverse primer, 2 μl of SYTO 9 fluorescent dye, and PCR-grade pure water were added to make the reaction volume 20 μl. Reaction at 95°C for 15 minutes, 95°C for 15 seconds, 60°C for 15 seconds, 72°C for 15 seconds for 20 cycles, 81°C for 15 seconds, 60°C for 15 seconds, 72°C for 15 seconds for 15 cycles, 72°C for 2 Minutes; af...

Embodiment 3

[0079] Example 3: Kit Sensitivity Verification

[0080] The A549 homozygous mutant genome was mixed with the wild-type genome extracted from whole blood in a certain proportion, so that the A549 genome accounted for 0.05% of the total DNA amount. The concentration of the above mixed genomic DNA and wild-type genomic DNA was adjusted to 10 ng per microliter.

[0081] . Specific primers are as follows:

[0082] SEQ ID No: 1, forward primer 1: 5'-GCCTGCTGAAAATGACTGAA-3'

[0083] SEQ ID No: 2, reverse primer 1: 5'-TATCGTCAAGGCACTCTTGC-3'

[0084] SEQ ID No: 7, specific probe: 5'-CTCTTGCCTACGCCACCAGCTCCAACT-3'.

[0085] . Amplify a fragment near the 12 / 13 codon by PCR; prepare a mixture: add 1 μl of the previously prepared genomic DNA solution, 2 μl of PCR buffer (10×), 1.6 μl of 2.5mM dNTP, 0.1 μl of HotStarTaq DNA polymerase, forward 0.4 μl of primer and 0.08 μl of reverse primer, 2 μl of SYTO 9 fluorescent dye, 200 nM of specific probe, and PCR-grade pure water were added ...

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Abstract

The invention relates to a method and kit for detecting gene mutations, particularly a method and kit for detecting KRAS gene 12, 13 codon mutations. The kit comprises a PCR (Polymerase Chain Reaction) buffer solution, a dNTP (deoxyribonucleotide triphosphate), a DNA (deoxyribonucleic acid) polymerase, a specific primer pair, fluorescent dye, water, a specific probe and a wild-type control. The kit is characterized in that the DNA polymerase is a HotStarTaq DNA polymerase, and the fluorescent dye is SYTO9 fluorescent dye. The method comprises the following steps: (1) acquiring a genome DNA to be analyzed according to a conventional method; (2) carrying out PCR amplification on the genome DNA to obtain a PCR amplified product; after the reaction finishes, carrying out denaturation and renaturation on the PCR product; and (3) carrying out melting curve analysis on the PCR amplified product, and comparing with a melting curve generated by the PCR amplified product of the wild-type genome DNA, wherein the melting curve generated by the PCR amplified product of the mutant genome DNA firstly descends.

Description

technical field [0001] The invention relates to a method and kit for detecting gene mutations, in particular to a method and kit for detecting mutations of 12 and 13 codons of KRAS gene. Background technique [0002] Genes are common oncogenes in human tumors. The RAS gene family consists of KRAS, HRAS and NRAS, and the homology among the members of the gene family can reach 85%. The protein encoded by the RAS gene is P21 protein, with a molecular weight of 21KD and composed of 188-189 amino acids, also known as P21 highly related protein. P21 protein is located on the inner surface of the cell membrane, has GTPase activity, and participates in the regulatory system of transmitting cell proliferation signals. Its activation state is GTP-binding state, and its inactivation state is GDP-binding state. The main parts of its transformation into an active oncogene are the mutations of the 12th, 13th and 61st codons, among which the point mutation of the 12th codon is the most c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 张泓姬云侯青
Owner 苏州科贝生物技术有限公司
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