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Method for detecting ARMS-ddPCR (amplification refractory mutation system-droplet digital polymerase chain reaction) gene site-directed mutation

A detection method and point mutation technology, applied in the field of gene point mutation detection methods, can solve the problems of long optimization time, small dynamic range, false positives, etc., and achieve the effects of high sensitivity, simple optimization and high specificity

Inactive Publication Date: 2019-04-09
江苏苏博生物医学科技南京有限公司 +1
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Problems solved by technology

Moreover, the position of the mutation site in the probe sequence is not fixed, requiring a large number of experimental optimizations, which takes a long time and costs high, and the dynamic range of its detection is small, which is prone to false positives

Method used

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  • Method for detecting ARMS-ddPCR (amplification refractory mutation system-droplet digital polymerase chain reaction) gene site-directed mutation
  • Method for detecting ARMS-ddPCR (amplification refractory mutation system-droplet digital polymerase chain reaction) gene site-directed mutation
  • Method for detecting ARMS-ddPCR (amplification refractory mutation system-droplet digital polymerase chain reaction) gene site-directed mutation

Examples

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Embodiment 1

[0025] Example 1 Detection of EGFR gene T790M mutation site

[0026] The human EGFR gene mainly produces mutations in four exons, which are exons 18, 19, 20 and 21, of which exon 18 has 5 mutant types, exon 19 has 34 mutant types, and exon 20 has mutated types. There are 9 mutant types in exon 21 and 3 mutant types in exon 21. In this example, the most common exon 20 T790M mutation of the EGFR gene was selected to compare the ARMS-ddPCR method of the present application with the existing ARMS-qPCR method and ddPCR method.

[0027] Construction of EGFR Gene T790M Mutation Site Plasmid Standards

[0028] Site-directed mutation was performed on the T790M site by Overlap PCR method, and the mutant gene was constructed into a plasmid.

[0029] The primers used by the Overlap PCR method are exon 20 primers and T790M site primers, as shown in Table 1.

[0030] Table 1 Construction of EGFR gene T790M mutation site primer list

[0031]

[0032] The 20-exon primers F and T790M R,...

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Abstract

The invention discloses a method for detecting ARMS-ddPCR (amplification refractory mutation system-droplet digital polymerase chain reaction) gene site-directed mutation. The method aims at gene mutation of one site, primers comprise a wild type primer and a mutation primer, probes comprise a wild type probe and at least one mutation probe, a mutation site is detected by further combination witha ddPCR platform, and sample mutation site DNA is subjected to absolute quantification. The method is particularly suitable for amplification of small-fragment DNA samples such as plasma free DNA (cfDNA) and the like, with application of the method, gene mutation with quite low abundance can be detected from cfDNA, the method has the characteristics of good specificity and high sensitivity, can beapplied to absolute quantification detection of the gene mutation site by the ddPCR platform and has a quite important effect on guidance of clinic treatment and improvement of patient outcome.

Description

technical field [0001] The invention relates to a method for detecting gene point mutations, belonging to the field of molecular biology, in particular to an ARMS-ddPCR gene mutation detection method formed by combining ARMS-qPCR and ddPCR. Background technique [0002] There are two commonly used PCR-based gene mutation detection methods: ARMS-PCR and digital PCR. ARMS-PCR: Amplification Refractory Mutation System PCR (Amplification Refractory Mutation System PCR, ARMS-PCR), also known as allele-specific PCR (Allele-Specific PCR, AS-PCR). ARMS-PCR technology is based on the allele-specific extension reaction, only when the 3' terminal base of an allele-specific primer is complementary to the base at the mutation site, the extension reaction can be carried out, and the template Incompletely matched upstream primers will not anneal and produce no PCR product. More than 90% of the currently popular on the market, such as tumor-targeted drug EGFR gene detection and CFDA-appro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6827
CPCC12Q1/6827C12Q2535/137C12Q2563/159C12Q2531/113
Inventor 苏雪峰张明航
Owner 江苏苏博生物医学科技南京有限公司
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