Rare mutation detection method, kit and application thereof

A detection kit and a technology for rare mutations, applied in the field of molecular biology, can solve the problems of different mismatch discrimination capabilities, only 20% sensitivity, and easy contamination of non-closed tubes, to achieve enhanced mutation base recognition capabilities, high sensitivity, The effect of reducing pollution

Inactive Publication Date: 2019-01-25
北京协和洛克生物技术有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has the disadvantages of long detection period, easy pollution in non-closed tube operation, low flux, and the sensitivity is only 20%.
The next-generation sequencing method is an emerging detection method, which has the advantages of high throughput and high sensitivity, and the sensitivity can reach 1%, but the detection cycle is longer, and mutations are easily introduced during the detection process
ARMS has a short detection cycle, closed-tube operation, high sensitivity, and a detection ability of 1%. However, ARMS primers have different ability to distinguish mismatches with different base pairs at the 3' end of the primer, so the ability to distinguish some mutations is limited.

Method used

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  • Rare mutation detection method, kit and application thereof
  • Rare mutation detection method, kit and application thereof
  • Rare mutation detection method, kit and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0067] According to the missense mutation at position 2369 of the EGFR gene, that is, the position 2369 is changed from C to T (T790M for short), the wild-type plasmid and the mutant-type plasmid were respectively constructed as templates through genetic engineering. The sequence of the wild-type plasmid is shown in SEQ ID No: 1. After determining the concentration of the plasmid, dilute it to 1ng / ul with TE buffer, and then carry out a 10-fold gradient dilution of the 1ng / ul plasmid with TE buffer, and the dilution concentration is 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 ng / ul spare, 10 -6 The gene copy number contained in ng plasmid is equivalent to the target gene copy number contained in 1ng genomic DNA.

[0068] The diluted mutant plasmid and wild-type plasmid were mixed in different proportions to obtain wild-type samples (without mutant plasmids), 1% mutant samples (the ratio of mutant plasmids to wild-type plasmids was 1:99), 5% Mutant samples (ratio of mutant p...

Embodiment 2

[0074] Using the mutant plasmid as a template, the nucleotide sequences of SEQ ID No:6 and SEQ ID No:2 are ARMS primers, the nucleotides shown in SEQ ID No:7 are universal primers and the nucleotides shown in SEQ ID No:8 Fluorescent PCR was carried out for the Taqman-MGB probe. In the fluorescent PCR reaction, the ratios of universal primers and AMRS upstream primers were 10:1, 20:1, 30:1, 40:1, 50:1, and the concentrations of the remaining components were the same ( The final concentration of each component in the 20μl system is 1×PCR Buffer Mix, 500nM ARMS downstream primer, 500nM universal primer, 250nM Taqman-MGB probe, 10 -6 ng mutant plasmid and balance water). Fluorescence PCR reaction condition is identical with embodiment 1, and result is as follows image 3 shown. Depend on image 3 It can be seen that when the ratio of universal primers to ARMS upstream primers is 40:1, the amplification efficiency is the best.

Embodiment 3

[0076] Adopt T790M site to carry out experiment, with the 100% mutant sample prepared in embodiment 1, 50% mutant sample, 20% mutant sample, 10% mutant sample, 5% mutant sample, 1% mutant sample and wild-type sample (adding amount is average for 10 -6 ng) as a template, using primers and primer ratios and Taqman-MGB probes in Example 2 to carry out fluorescent PCR to verify its sensitivity, the results are as follows Figure 4 shown. Depend on Figure 4 It can be seen that the detection sensitivity of the detection method disclosed in the present invention can reach 1%.

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Abstract

The invention discloses a rare mutation detection method, a kit and an application thereof. The method is an ARMS method. The detection method provided by the invention combines ARMS and locking nucleic acid (LNA) mutation enrichment and Taqman-MGB fluorescence detection technology, using LNA to enhance the ability of base recognition, aRMS primers to mutation target sequence specific PCR amplification, a taqman-MGB probe detects that amplification product and identifies a specific mutation on the basis of fluorescent PCR. Compared with mutation detection technologies such as sanger sequence,chip detection and second generation sequencing, the method has the advantages of low cost, strong specificity, high sensitivity, simple and rapid operation and the like for gene mutation detection.

Description

technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to a rare mutation detection method, a kit and application thereof. Background technique [0002] Polymerase chain reaction (PCR) was invented in 1985 by Professor Kary B Mullis of the Department of Genetics of PE Company in the United States, which brought a major leap forward in gene amplification technology. Because this technology can specifically amplify the target nucleic acid sequence to several million times through 20-50 cycles consisting of three steps of denaturation, annealing and extension within 1.5-3 hours, therefore, in just a few years, molecular It is widely used in biology, medicine, microbiology, genetics and other fields. [0003] Nucleic acid is the key substance that determines genetic information. With the continuous development of life science research, people have found that by detecting whether there is a certain (some) nucleic acid and its seq...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2531/113C12Q2535/137C12Q2561/101
Inventor 刘永吴建榕
Owner 北京协和洛克生物技术有限责任公司
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