Rapid detection method of mutation of PTEN gene

A detection method, PTEN-E5 technology, applied in the fields of biotechnology and medicine, can solve the problems of undetectable low-ratio mutations, low sensitivity, and high cost, and achieve the effects of high cost, high sensitivity, and low single cost

Active Publication Date: 2013-11-20
山东国九堂制药集团股份有限公司
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AI Technical Summary

Problems solved by technology

[0021] First: the sensitivity is not high, and a low proportion of mutations (<20%) cannot be detected;
[0022] Second: it takes a long time, usually 2-3 days;
[0023] Third: high cost

Method used

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  • Rapid detection method of mutation of PTEN gene
  • Rapid detection method of mutation of PTEN gene
  • Rapid detection method of mutation of PTEN gene

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Experimental program
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Embodiment Construction

[0065] The invention is applied to the fields of biotechnology and medicine, especially molecular biology, molecular diagnosis and real-time quantitative PCR technology.

[0066] Such as Figure 1-3 As shown, we designed wild-type primers and mutant primers for PTEN exon 5 (PTEN-E5) and exon 6 (PTEN-E6), respectively, to amplify the corresponding wild-type target fragment and mutant For the target fragment, mix the two fragments in different proportions so that the content of the mutant target fragment is 1 / 10, 1 / 100, 1 / 1000 and 0, and finally use the HRM method to distinguish. The following takes PTEN-E2 as an example to illustrate the specific operation.

[0067] 1. Sample processing: take 2ml of peripheral blood into EDTA anticoagulant tubes, gently invert and mix, and store at 4°C.

[0068] 2. DNA extraction: Take 200 μL of anticoagulated blood and extract DNA with QIAmp DNA Blood Mini Kit kit. The purity and concentration of DNA were detected by electrophoresis gel ima...

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Abstract

The invention provides a rapid detection method of the mutation of a PTEN (phosphatase and tensin homolog) gene. The method comprises the following steps: designing a pair of wild primers at two ends of the mutation site of the PTEN gene, and designing a pair of mutant primers on the mutation site; respectively designing the wild primers and the mutant primers of the PETN exon 5 (PTEN-E5) and the PETN exon 6 (PTEN-E6), amplifying the primers to obtain wild templates and mutant templates, and carrying out PCR amplification of the two templates through using the wild primers to respectively amplify corresponding wild target fragments and mutant target fragments; and mixing the above two fragments according to different proportions, and distinguishing through using an HRM process. Compared with other methods, the method provided by the invention has the advantages of high specificity, high sensitivity, convenience, fastness, high flux and low single cost.

Description

Technical field: [0001] The invention relates to the fields of biotechnology and medicine, especially molecular biology, molecular diagnosis and real-time quantitative PCR technology. Background technique: [0002] The PTEN gene is located in the 10q23.3 region of human chromosome, contains 9 exons, has a total length of 1212bp, encodes a protein consisting of 403 amino acid residues, and the relative molecular weight of the product is 56kDa. [0003] PTEN is a tumor suppressor gene, which inhibits tumor production through various pathways. The wild-type PTEN protein mainly has the following functions: ① Participate in the normal development of embryos. ② PTEN and phosphatidylinositol 3-kinase (phosphatidyl inositol 3-kinase, PI3K) coordinated effect, so that the concentration of PIP3 in the cell is at a moderate level, maintain the activity of protein kinase B (AKT), and promote cell apoptosis. ③ PTEN can regulate a variety of cell cycle-related proteins and regulate the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王明彭南求
Owner 山东国九堂制药集团股份有限公司
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