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Multiple cancer risk susceptibility mutation site, application thereof and produced kit thereof

A mutation site and susceptibility technology, applied in the field of tumor molecular genetics, can solve the problems of inappropriateness and high cost, achieve good sensitivity and improve the effect of discriminating efficiency

Inactive Publication Date: 2012-01-11
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

DNA chip technology is a newly developed DNA sequence variation detection tool in recent years. However, if there are not many mutation sites involved, high-throughput DNA chips are not suitable due to high cost.

Method used

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  • Multiple cancer risk susceptibility mutation site, application thereof and produced kit thereof
  • Multiple cancer risk susceptibility mutation site, application thereof and produced kit thereof
  • Multiple cancer risk susceptibility mutation site, application thereof and produced kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preliminary studies have found that 3q24-26 is closely linked to multi-cancer families

[0030] In the previous research, the inventor discovered a rare multi-cancer family in Hunan, with more than 103 people in six generations. Among them, there were 15 patients, and 10 survived, involving a variety of benign and malignant tumors. For the family map of multiple cancers, see figure 1. In order to locate the chromosome segment linked to the disease, we first used a high-throughput SNP chip (Human Mapping 500K SNP Array of Affymetrix Company) to scan and type the whole genome, and performed single-point parameter linkage analysis through Merlin software. The area with LOD value greater than 2 is obtained. Later, microsatellites were used for fine positioning in this area. When multi-point non-parametric linkage analysis was performed by Genehunter software, the maximum LOD value reached 10.674 (p=0.00195), which was located between D3S1584 and D3S3710, confir...

Embodiment 2

[0032] Example 2: Preparation of Tumor Risk Susceptibility Mutation Screening Kit

[0033] (1) Primer design for the mutation site

[0034] 1. Selection of template sequence

[0035] The template sequence was from the UCSC database ( http: / / genome.ucsc.edu / ), select 300 bp upstream and downstream of the mutation site, to ensure that the PCR product is in a suitable fragment range for easy detection.

[0036] 2. Primer Design

[0037] There are 3 basic principles for PCR primer design: firstly, primers should be closely combined with the template, secondly, there should be no stable dimer or hairpin structure between primers, and thirdly, primers should not cause DNA polymerization at other non-target sites (i.e. mismatch). Specific considerations include: primer length, product length, sequence Tm value (melting temperature), ΔG value (internal stability), primer dimer and hairpin structure (duplex formation and hairpin), error Initiation site (false priming site), prim...

Embodiment 3

[0070] Embodiment 3: The steps of using the mutation screening kit of the present invention

[0071] Step 1 Blood sample collection and gDNA extraction

[0072] Collection and processing of blood samples: using the German Greiner company (Non-replaceable) 5ml EDTA anticoagulant tube, collect 5ml of peripheral blood sample from the individual to be tested, store at 4°C, and extract gDNA within two weeks. If the gDNA is not extracted immediately, the anticoagulant tube can be stored in the refrigerator at -20°C, and extracted together after collecting multiple samples.

[0073] gDNA extraction: Blood gDNA was extracted using the TaKaRa Whole Blood Genome Extraction Kit (Universal Genomic DNA Extraction Kit Ver.3.0) (see the product manual for details). Take 2 μl DNA sample to run on the gel, and take 2 μl gDNA sample to dilute to 100 μl at the same time, measure the absorbance value, and calculate the extracted DNA concentration according to the formula: DNA concentration = a...

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Abstract

The invention discloses a multiple cancer risk susceptibility mutation site, an application thereof and a produced kit thereof. The research verifies that a multiple cancer family chromosome 3q24-26 area is tightly linked with a disease site, gene mutation screening verifies that ATG upstream-91bp of human TSC22D2 gene T>C mutation (c.-91T>C) is co-separated from pathotype in a multiple cancer family, According to large scale sequencing verification in local normal population, the mutation is negative. The genotype provides that the mutation site can be used for screening the tumor heredity susceptible population and predicting the prevalence risk of tumor. The invention also provides a kit for screening the relative mutation. The kit has the advantages of high sensitivity, good specificity, high flux and the like. Compared with the traditional sequencing method, a PCR product is not required for purifying, and the method is capable of minimizing the interference of artificial factors like cross-contamination, simultaneously the labor resources, the financial resources and the material resources can be saved.

Description

technical field [0001] The invention belongs to the field of tumor molecular genetics, and relates to a multi-cancer risk susceptibility mutation site and a kit for its application and preparation. Background technique [0002] Tumor is a polygenic complex disease, and its occurrence is the result of the joint action of environmental factors and tumor genetic susceptibility factors. Gene mutation is an important reason for the differences in the genetic susceptibility of different individuals to tumors. Mutations of oncogenes and tumor suppressor genes, mitochondrial DNA mutations, and dynamic mutations of unstable trinucleotide repeat sequences are all related to the genetic susceptibility of tumors. If the mutations of oncogenes and tumor suppressor genes are clustered in families and populations, which can significantly increase the risk of cancer, then screening for these mutations in healthy or tumor populations will undoubtedly help people with genetic susceptibility ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 李桂源肖岚熊炜曾朝阳谭世明
Owner CENT SOUTH UNIV
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