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Rapid detection method of gene mutation of Caveolin-1

A detection method and a rapid technology, applied in the fields of biotechnology and medicine, can solve the problems of undetectable low proportion of mutations, low sensitivity, high cost, etc., and achieve the effect of high cost, high sensitivity, and low single cost

Inactive Publication Date: 2015-06-03
南京赛安生物医药科技有限公司
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0007] First: the sensitivity is not high, and a low proportion of mutations (<20%) cannot be detected;
[0008] Second: it takes a long time, usually 2-3 days;
[0009] Third: high cost

Method used

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  • Rapid detection method of gene mutation of Caveolin-1
  • Rapid detection method of gene mutation of Caveolin-1
  • Rapid detection method of gene mutation of Caveolin-1

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Experimental program
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Embodiment Construction

[0044] The invention is applied to the fields of biotechnology and medicine, especially molecular biology, molecular diagnosis and real-time quantitative PCR technology.

[0045] As shown in the figure, the present invention designs wild-type primers and mutant primers of Caveolin-1 to amplify corresponding wild-type target fragments and mutant-type target fragments respectively, and mix these two fragments in different proportions to make the mutant-type target fragments The content is 1 / 10, 1 / 100, 1 / 1000 and 0, and finally the HRM method is used to distinguish. The following takes Caveolin-1P132L as an example to illustrate the specific operation.

[0046] 1. Sample processing: take 2ml of peripheral blood into EDT1A anticoagulant tube, gently invert and mix, and store at 4°C.

[0047] 2. DNA extraction: Take 200 μL of anticoagulated blood and extract DNA with QIAmp DNA Blood Mini Kit kit. DNA purity and concentration were detected by electrophoresis gel imaging.

[0048]...

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Abstract

The invention provides a rapid detection method of gene mutation of Caveolin-1. The method comprises the following steps: designing a pair of wild primers at both ends of a gene mutation site of Caveolin-1, and designing a pair of mutation primers at the mutation site; respectively amplifying wild templates and mutant fragments, and carrying out PCR (Polymerase Chain Reaction) amplification on two mutant fragments by the wild primers to amplify corresponding mutant templates; and mixing the wild templates and mutant templates in different proportion, and differentiating by an HRM (High Resolution Melting) method. Compared with other methods, the method provided by the invention has the advantages of high specificity, high sensitivity and convenience, and is higher in flux and lower in single-time 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] Caveolin-1 gene is located on human 7q31.1 and contains 3 exons. Caveolin-1 in mammalian cells can be divided into α-type and β-type (Caveolin-1α and Caveolin-1β) according to the difference of N-terminus. The main difference between them is that DNA is transcribed into mRNA from different starting points. Caveolin-1α is 24kDa and consists of 178 amino acid residues; Caveolin-1β is 21kDa and consists of 147 amino acid residues. [0003] Caveolin-1 inhibits tumorigenesis by regulating the cell cycle. After Caveolin-1 is normally expressed, its N-terminus binds to the promoter of CyclinD1 gene to inhibit the activity of CyclinDl. Caveolin-1 can sequester the phosphorylated tyrosine protein kinase Fyn (synchronous proto-oncogene) and SH2 carrying protein (Src homo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2527/107C12Q2531/113
Inventor 王明彭南求
Owner 南京赛安生物医药科技有限公司
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