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Mismatch repair gene MLH1 mutation detection kit and application thereof

A detection kit and mismatch repair technology, applied in the field of mutation consequence assessment and genetic diagnosis, can solve the problems of lack of effective evaluation methods for missense mutations or synonymous mutations

Inactive Publication Date: 2011-02-16
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of effective mutation detection methods for East Asian populations, systematic screening and case-control analysis of this gene mutation have not been reported in China
There is also a lack of effective means of assessing the functional consequences of detected missense or synonymous mutations
In particular, the evaluation of the consequences of MLH1 gene mutations from the perspective of RNA splicing has not been reported.

Method used

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  • Mismatch repair gene MLH1 mutation detection kit and application thereof
  • Mismatch repair gene MLH1 mutation detection kit and application thereof
  • Mismatch repair gene MLH1 mutation detection kit and application thereof

Examples

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Effect test

Embodiment 1

[0028] Screening and consequence assessment of exon 12 c.1151T>A(V384D) mutation of MLH1 gene: 236 patients with gastric cancer in Jiangsu, Anhui and Zhejiang regions of China, and 240 normal controls were selected, peripheral blood was drawn, and DNA was extracted. For the 12th exon of MLH1 gene, PCR amplification and HRM analysis were performed according to the primers in Table 1. The reaction system is: Genomic DNA (100ng / μl) 1.0μl, dNTP Mixture (2.5mM each) 0.8μl, 10×PCR buffer (Mg 2+ free) 1.0μl, MgCl 2 (25mM) 1.0μl, upstream primer (10μM) 0.2μl, downstream primer (10μM) 0.2μl, dimethyl sulfoxide (DMSO) 0.4μl, ddH 2 O4.32μl, LC Green dye 1.0μl, Taq DNA polymerase (5Unit / μl) 0.08μl. Reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, renaturation at 60°C for 30 sec, extension at 72°C for 40 sec, 40 cycles; extension at 72°C for 7 min, and storage at 4°C. HRM instrument LightScanner 96 (Idaho, USA) detection: open the Lightscanner so...

Embodiment 2

[0038] Screening and consequence assessment of exon 18c.2101C>A(Q701K) mutation of MLH1 gene: 236 patients with gastric cancer in Jiangsu, Anhui and Zhejiang regions of China, and 240 normal controls were selected, peripheral blood was drawn, and DNA was extracted. For exon 18 of MLH1 gene, primers were designed according to Table 1 for PCR amplification and RFLP analysis. The reaction system is: Genomic DNA (100ng / μl) 1.0μl, dNTP Mixture (2.5mM each) 0.8μl, 10×PCR buffer (Mg 2+ free) 1.0μl, MgCl 2 (25mM) 1.0μl, upstream primer (10μM) 0.2μl, downstream primer (10μM) 0.2μl, dimethyl sulfoxide (DMSO) 0.4μl, ddH 2 O 5.32 μl, Taq DNA polymerase (5 Unit / μl) 0.08 μl. Reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, renaturation at 56°C for 30 sec, extension at 72°C for 40 sec, 40 cycles; extension at 72°C for 7 min, and storage at 4°C. Restriction fragment length polymorphism (RFLP) analysis: looking for specific restriction endonuclease C...

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Abstract

The invention relates to a mismatch repair gene MLH1 mutation detection kit and application thereof. The kit comprises the main components: firstly, 20 pairs of MLH1 gene PCR (Polymerase Chain Reaction) amplification and sequencing primers; and secondly, a reagent for PCR amplification. A PCR amplification product obtained through the kit can be used for screening gene micromutation through a high resolution dissolution curve method or carrying out restriction fragment length polymorphism analysis. The kit has high efficient MLH1 gene mutation detection and function evaluation actions and is used for mismatch repair gene MLH1 mutation detection.

Description

technical field [0001] The invention relates to a mismatch repair gene MLH1 mutant detection kit and its application, in particular to its gene diagnosis and mutation consequence evaluation in East Asian gastric cancer high-incidence population. Background technique [0002] Gastric cancer (GC) is the third most common malignant tumor in my country, with an annual mortality rate as high as 25 / 100,000. Among them, the suspected hereditary cases were greater than 20%, suggesting that genetic factors played an important role in the pathogenesis of gastric cancer, but the detection rate of mutations was not high. It is known that the MLH1 gene encodes a protein that plays a central role in mismatch repair during DNA replication. The MLH1 gene contains 19 exons, encoding 754 amino acids. The conserved amino-terminus of MLH1 protein is considered to be related to ATP bonding, while the carboxy-terminal region is considered to interact with PMS2 protein. Mutations in the MLH1 ge...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 范怡梅支文贤薛冰霜陈锦云张婉芬郭文文王亚平
Owner NANJING UNIV
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