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Digital PCR platform based gene deletion mutation detection method and kit thereof

A gene deletion and kit technology, applied in the field of molecular biology, can solve problems such as poor sensitivity and inability to meet high-sensitivity detection requirements, and achieve the effect of improving detection sensitivity

Inactive Publication Date: 2014-07-16
上海涌泰生物医药科技有限公司
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Problems solved by technology

[0010]3. The sensitivity is relatively poor. The sensitivity of the current method is best at 1%, which cannot meet some high-sensitivity detection requirements

Method used

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  • Digital PCR platform based gene deletion mutation detection method and kit thereof

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Embodiment 1

[0044] (1) Prepare DNA samples to be tested: DNA containing wild-type EGFR gene, EGFR exon 19 deletion mutation-positive DNA mixed with wild-type DNA (the ratio of mutant to wild-type content is 1 / 1, 1 / 100, 1 / 1000, 1 / 2500). The source of DNA can be serum, plasma, peripheral blood, oral mucosa, pleural effusion, body fluid or tissue, etc. The mutant DNA was derived from a positive cell line carrying the EGFR19 mutation. Among them, the c.2238-c.2255 deletion mutation on the EGFR19 exon.

[0045] (2) Melt forward and reverse detection PCR amplification primers and corresponding labeled TaqMan probes and PNA probes for detecting EGFR exon 19 deletion at room temperature, as well as forward and reverse quality control PCR primers and quality control PCR primers. control TaqMan probes.

[0046] The sequences of forward and reverse PCR amplification primers are:

[0047] F1: 5'-GAGAAAGTTAAAATTCCCGTCGCT-3'

[0048] R1:5'-ATGGACCCCCCACACAGCA-3'

[0049] The sequence of the label...

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Abstract

The invention relates to the field of the molecular biology, and concretely relates to gene deletion mutation detection method and a kit thereof. A PNA probe complementary to a wild non-mutation template sequence is added into a reaction system with digital PCR as a platform, and the repression effect of the PNA probe is utilized to make only deletion-mutated samples amplified; and the existence of mutant templates and the quantity and the proportion of the mutant templates are determined through fluorescence detection by utilizing a TaqMan probe as a fluorescent quantitative mark.

Description

technical field [0001] The invention relates to the fields of molecular biology and nucleic acid detection, in particular to a method for detecting gene deletion mutations. Background technique [0002] Deletion mutation is a common type of gene variation, which will bring many changes to the biological traits of microorganisms, plants and animals, and the physiological traits of humans; including partial or complete inactivation of protein functions, formation of inactive fragments, etc., and may also change biological traits. Gene regulation and other functions of the body. The study of gene mutation is of great significance to the inheritance, evolution and transformation of organisms. [0003] At present, there are mainly direct sequencing method (also known as Sanger sequencing method) and ARMS method for detection of gene variation. They are briefly introduced as follows: [0004] The Sanger sequencing method, that is, the Sanger (Sanger) dideoxy chain termination m...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2561/101C12Q2537/163C12Q2563/159
Inventor 王世亨
Owner 上海涌泰生物医药科技有限公司
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