Method for detecting colorectal cancer driving gene and susceptible SNP (Single Nucleotide Polymorphism) based on nucleic acid mass-spectrometric technology

A technology for colorectal cancer and driver genes, which is applied in the field of detecting colorectal cancer driver genes and their susceptibility SNPs based on nucleic acid mass spectrometry technology, can solve the problems of insufficient flexibility, low detection accuracy, and small detection capacity.

Inactive Publication Date: 2018-01-09
武汉赛云博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the commonly used methods for detecting SNPs mainly use TaqMan-PCR and its second-generation platform s...

Method used

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  • Method for detecting colorectal cancer driving gene and susceptible SNP (Single Nucleotide Polymorphism) based on nucleic acid mass-spectrometric technology
  • Method for detecting colorectal cancer driving gene and susceptible SNP (Single Nucleotide Polymorphism) based on nucleic acid mass-spectrometric technology
  • Method for detecting colorectal cancer driving gene and susceptible SNP (Single Nucleotide Polymorphism) based on nucleic acid mass-spectrometric technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Feasibility analysis of screening SNPs of colorectal cancer susceptibility-related genes and hotspot mutation sites of rectal cancer driver genes

[0031] The inventors of the present invention searched NCBI domestic and foreign genome-wide association study (genome-wide association study, GWAS) research documents and related patents (patent documents 1-3, non-patent documents 1-9) through sequencing and analysis of the whole genome of colorectal cancer ), searched the GWAS Catalog database and cancer-related databases such as COSMIC, SNPedia, UCSC, ICGC, and MY CANCERGENOME, and screened out more than 1,000 positive samples from the Asian population and more than 500 positive samples from the Chinese population in the GWAS study. The inventors screened and evaluated the sites that were significantly associated with the occurrence of CRC that were verified in clinical studies, and selected 25 CRC hotspot mutations and 22 single nucleotide polynucleotides that ...

Embodiment 2

[0049] Example 2: Detection of colorectal cancer susceptibility-related SNP sites and CRC hotspot mutation sites by the technology of the present invention

[0050] 1. Extract DNA from peripheral blood samples to obtain DNA extraction solution;

[0051] 1. Take 200μl peripheral blood sample and put it into a 1.5ml centrifuge tube. (If the initial volume of whole blood is less than 200μl, use buffer BB to make up to 200μl. If the initial volume is between 200μl-300μl, then increase the reagent volume proportionally in subsequent operations. If the initial volume is between 300μl-1ml lysing of red blood cells is required first)

[0052] 2. Add 200 μl of binding solution CB, shake it upside down vigorously immediately, and mix well, then add 20 μl proteinase K (20 mg / ml) solution, shake it upside down and mix well, place at 70°C for 10 minutes, the solution should become clear.

[0053] 3. Add 100 μl of isopropanol, shake vigorously upside down, and mix thoroughly. At this time...

Embodiment 3

[0106] Example 3: Detection of colorectal cancer cell lines using the method of Example 2

[0107] 1. Detection object

[0108] Select T84, HCT116, HT-29, LS174T, SW620, MOLT-46 colorectal cancer cell lines with known mutation sites (all can be purchased from ATCC) for feasibility analysis of the detection method, and set up normal human genomic DNA (gDNA) (extracted from oral mucosal tissues of normal people) was used as a negative control, and water was used as a blank control.

[0109] 2. Experimental steps

[0110] The concrete operation method that the technology of the present invention is used for this embodiment is as follows:

[0111] (1) extracting the DNA of the colorectal cancer cell line to obtain a DNA extract;

[0112] (2) PCR amplification; the amplification primer mixture in Example 2 is used as the amplification primer, and the DNA extract is used as the amplification template to carry out PCR amplification reaction to obtain the PCR product fragment of th...

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Abstract

The invention relates to the field of gene detection, in particular to a method for detecting colorectal cancer driving genes and susceptible SNPs based on nucleic acid mass spectrometry technology. The present invention selects 25 CRC hot spot mutation sites and 22 SNP sites that are significantly related to the risk of lung cancer in Asian populations and have independent functions by analyzing the whole genome sequence of colorectal cancer and analyzing the positive samples of Asian population in GWAS research. To assess the risk of an individual developing CRC. Compared with TaqMan-PCR and second-generation platform sequencing, the detection method has the characteristics of simplicity, flexibility, high throughput, low cost, and the like. In addition to being used for early assessment and large-scale screening of CRC, the present invention provides an important reference basis for risk assessment and diagnosis of CRC, so as to realize early warning of colorectal cancer disease, and at the same time, according to the detection results, CRC patients can be assessed. Personalized treatment.

Description

technical field [0001] The invention belongs to the field of gene detection, in particular to a method for detecting colorectal cancer driver genes and their susceptible SNPs based on nucleic acid mass spectrometry Background technique [0002] Colorectal cancer (CRC) is one of the most common malignant tumors in the world. In the past decade, the morbidity and mortality of CRC have risen sharply, and it has become a significant public health problem in China. Although the treatment of colorectal cancer has made great progress in recent years, the overall curative effect has not been greatly improved, and the 5-year survival rate after surgery is about 50%. The treatment effect of early colorectal cancer patients is better, and the 5-year survival rate can be above 90%. The factors leading to the pathogenesis of CRC mainly include environment and genetics. Many environmental factors such as nutrition, lack of exercise, obesity, smoking and alcohol consumption are the etiol...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/6886
Inventor 雷菁赵双双尚小云张轶刁波
Owner 武汉赛云博生物科技有限公司
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