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Method of detecting gene mutation based on Blocker primers and ARMS primers, and kit

A kit and mutant technology, applied in the field of molecular biology, can solve the problems of inability to effectively distinguish mutant templates from wild-type templates, reduce wild-type blocking efficiency, and mutant detection efficiency

Active Publication Date: 2013-08-21
北京宏微特斯生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is also a problem with this method. During the blocking process of Blocker primers, Blocker cannot effectively distinguish mutant templates from wild-type templates. Although there is a base difference (for the mutation site), the annealing temperature of the primers The lower (melting temperature, Tm) Blocker primer can still be combined with the mutant template, or the primer can also be combined with the wild-type template, thereby reducing the efficiency of wild-type blocking and the detection efficiency of the mutant

Method used

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  • Method of detecting gene mutation based on Blocker primers and ARMS primers, and kit
  • Method of detecting gene mutation based on Blocker primers and ARMS primers, and kit
  • Method of detecting gene mutation based on Blocker primers and ARMS primers, and kit

Examples

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Effect test

Embodiment 1

[0072] Example 1 One-step detection method of ARMS fluorescence quantitative PCR for mutation blocking enrichment of EGFR gene point mutation T790M

[0073] 1. Primer and Probe Design

[0074] 1) Blocker primer design

[0075] Primers were designed near the mutation site (including the mutation site), and the 3' end was modified with dideoxy bases to prevent its extension and increase its annealing temperature. The relevant parameters are: Tm value 63.0°C-80.0°C, GC value 40.0%-65.0%, primer size 25±10bp.

[0076] The designed Blocker primer sequences are as follows:

[0077] Blocker primer: 5'-TGCAGCTCATCACGCAGCTCATG-3'ddC (SEQ ID NO: 1)

[0078] 2) ARMS primer design

[0079] Design primers that can amplify a 60-150bp fragment, the relevant parameters are: Tm value 55.0°C-60.0°C, GC value 40.0%-60.0%, primer size 20±3bp. The 3' end of the upstream ARMS primer is located at the mutation site and is consistent with the mutant gene. In order to improve the specificity, a m...

Embodiment 2

[0098] Example 2 EGFR gene exon 19 deletion E746_A750 deletion mutation mutation blocking enrichment ARMS fluorescent quantitative PCR one-step detection method

[0099] 1. Primer and Probe Design

[0100] 1) Blocker primer design

[0101] Primers were designed near the deletion mutation region (including the deletion sequence), and the 3' end was modified with dideoxy bases to prevent its extension and increase its annealing temperature. The relevant parameters are: Tm value 65.0°C-80.0°C, GC value 40.0%-65.0%, primer size 25±10bp.

[0102] The designed Blocker primer sequences are as follows:

[0103] Blocker primer: 5'-CAAGGAATTAAGAGAAGCAACATC-3'ddC (SEQ ID NO: 9)

[0104] 2) ARMS primer design

[0105] Design primers that can amplify a 60-150bp fragment, the relevant parameters are: Tm value 55.0°C-60.0°C, GC value 40.0%-60.0%, primer size 20±3bp. The 3' end of the upstream ARMS primer is located at the mutation site and is consistent with the mutant gene. In order to...

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Abstract

The invention discloses a method of detecting gene mutation based on Blocker primers and ARMS primers, and a kit. According to the invention, corresponding Blocker primers are designed according to an annealing temperature, the Blocker primers are combined with a wild-type template under the annealing temperature of the Blocker to block amplification of the wild type, and simultaneously an ARMS technology is used to reduce amplification of the wild-type background. The method integrates a Blocker enrichment technology and an ARMS fluorescent quantification PCR technology in one reaction system, and thus realizes enrichment and identification for the mutated genes for one step, reduces operation steps, and improves detection sensitivity and singularity.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method and a kit for detecting gene mutations. Background technique [0002] Gene mutation is a change in gene structure caused by the addition, deletion or change of base pairs in DNA molecules. There are two types of genetic mutations, sex cell mutations and somatic mutations. Sex cell mutation refers to a mutation that occurs in sex cells and is a type of heritable mutation. Mutations in somatic cells other than sex cells will not cause genetic changes in offspring, but can cause changes in the genetic structure of some contemporary cells. Most somatic mutations have no phenotypic effect. Somatic mutation is a kind of rare mutation. Somatic mutation exists in a large number of wild-type background DNA sequences. Compared with the content of wild-type background sequence, the content of somatic mutation is very small. For example, there is a small amount of tumo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6827C12Q1/6858C12Q2535/137C12Q2563/107C12Q2545/114C12Q2531/113
Inventor 陈唯军刘利成赵金银冯华华吴伟力王鹏志刘琦徐磊
Owner 北京宏微特斯生物科技有限公司
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