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MMR gene mutation detection kit

A kit and gene mutation technology, applied in the fields of molecular biology and medicine, can solve the problems of inability to meet the detection needs, time-consuming sequencing, and high price.

Inactive Publication Date: 2017-02-01
SHANGHAI CENT FOR BIOINFORMATION TECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Direct gene sequencing is the most sensitive and specific method for the detection of germline mutations of MMR genes, but sequencing based on the Sanger method is time-consuming and expensive, and cannot meet the detection needs of large clinical samples

Method used

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  • MMR gene mutation detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1 Primer Design and Synthesis

[0116] The present invention designs 87 pairs of Primers for a total of 11,419 bases in 73 exons of 5 genes (MSH1, MLH2, MSH6, PMS1, PMS2), and the length of the PCR amplification product is 257-528bp. The combined probe primer can Covering all exon regions of 5 MMR genes (see Table 1). Primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd. See Table 2 for primer sequence information.

[0117] Table 1: Reference sequence information table for primer design

[0118]

[0119] Table 2: 87 pairs of primers targeting 5 MMR genes

[0120]

[0121]

[0122]

[0123]

Embodiment 2

[0124] Example 2 Primer combination and multiplex PCR targeted enrichment method

[0125] 1. Experimental materials

[0126] (1), the main reagents

[0127] 1. Fluorescence quantitative detection kit: Qubit TM dsDNA HS Assay Kit (500 assays), Life technologies company;

[0128] 2. Quantitative detection kit: Agilent DNA 7500 and 12000Kit, Agilent Company, the kit includes;

[0129] 3. Multiplex PCR kit: MixMultiplex PCR 5×Master Mix, NEB Company;

[0130] 4. Sequencing library ligation adapter kit: NEXTflex TM DNA Barcode-48, BIOO SCIENTIFIC;

[0131] 5. Sequencing library construction kit: NEXTflex TM Rapid DNA-Seq Kit, BIOO SCIENTIFIC;

[0132] 6. Sequencing kit: MiSeq Reagent Kit v3 (600-cycles), Illumina

[0133] 7. Purification kit: Agencourt AMPure XP beads, Beckman Coulter (Agencourt) company

[0134] (2) Main instruments

[0135] 1. 0.5ul~2ml pipette, Eppendof, Germany;

[0136] 2. Micro centrifuge, Hangzhou Aosheng Instrument Co., Ltd.;

[0137] 3. High...

Embodiment 3

[0249] Example 3 Detection of MMR gene mutation in 17 cases of colorectal cancer and 14 normal persons

[0250] One, experimental material, with embodiment 2.

[0251] 2. Sample testing

[0252] (1) Multiplex PCR targeted enrichment

[0253] Genomic DNA was extracted from 31 samples (17 cases of colorectal cancer and 14 cases of normal people) using Qiagen's kit and according to the method in the kit instructions. The genomic DNA of 31 samples was amplified by multiplex PCR using 11 sets of primer pairs as shown in Table 3 of Example 2, and the specific method was the same as in Example 2.

[0254] (2), multiple PCR product purification, specific method is the same as embodiment 2.

[0255] (3), sequencing library construction, specific method is the same as embodiment 2.

[0256] (4), the quality inspection of the sequencing library, the specific method is the same as that in Example 2.

[0257] 3. High-throughput sequencing, the specific method is the same as that in Ex...

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Abstract

The invention belongs to the field of molecular biology and medicine, and particularly relates to a DNA mismatch repair gene (MMR) mutation detection kit and uses thereof, more particularly to mutation detection of human MLH1 gene, human MSH2 gene, human MSH6 gene, human PMS1 gene and human PMS2 gene, and detection of the correlation with hereditary nonpolyposis colorectal cancer (HNPCC). According to the present invention, the kit can be used for detecting the genotype of the exon mutation of the individual MMR gene so as to provide the reference data for judging whether the individual has HNPCC and whether the suffering risk of family members is greater than the suffering risk of the general populations.

Description

technical field [0001] The invention relates to the fields of molecular biology and medicine, in particular to a MMR gene mutation detection kit. Background technique [0002] Colorectal cancer (CRC) is the third most common malignant tumor in the world. Studies have found that 35% of CRC have familial susceptibility, the most common of which is hereditary nonpolyposis colorectal cancer (HNPCC), accounting for 2% to 5% of all cases. Germline mutations in DNA mismatch repair genes (MMR) are the genetic pathogenesis of Lynch syndrome. Studies have found that germline mutations of at least 5 MMR genes (MSH1, MLH2, MSH6, PMS1, PMS2) can cause HNPCC, of ​​which germline mutations in MLH1 account for 50%, MSH2 accounts for 40%, MSH6 accounts for 7%, and the remaining genes 3%. In the family, MMR gene germline mutation carriers are at high risk of HNPCC-related tumors, and the detection of MMR gene germline mutations can well predict the risk of HNPCC-related tumors. For patien...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/10
Inventor 黄凯李轩南蓬
Owner SHANGHAI CENT FOR BIOINFORMATION TECH
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