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Rapid detection method for p53 gene mutation

A detection method, p53-e6 technology, applied in the fields of biotechnology and medicine, can solve the problems of low sensitivity and undetectable low-ratio mutations, and achieve the effects of high sensitivity, high cost and high throughput

Active Publication Date: 2013-12-11
山东国九堂制药集团股份有限公司
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Problems solved by technology

[0016] The technical problems to be solved by the present invention are mainly: the existing sequencing method has three disadvantages: first: the sensitivity is not high, and a low proportion of mutations (<20%) cannot be detected;

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  • Rapid detection method for p53 gene mutation
  • Rapid detection method for p53 gene mutation
  • Rapid detection method for p53 gene mutation

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Embodiment Construction

[0043] The invention is applied to the fields of biotechnology and medicine, especially molecular biology, molecular diagnosis and real-time quantitative PCR technology.

[0044] Such as Figure 1-3 As shown, the present invention designs wild-type primers and mutant primers for exon 6 (p53-E6) and exon 8 (p53-E8) of p53 respectively to amplify the corresponding wild-type target fragment and mutation 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 p53-E6 as an example to illustrate the specific operation.

[0045] 1. Sample processing: take 2ml of peripheral blood into EDTA anticoagulant tubes, gently invert and mix, and store at 4°C.

[0046] 2. DNA extraction: Take 200 μL of anticoagulant blood and extract DNA with QI Amp DNA Blood Mini Kit kit. DNA purity and concentration were detected by electrophoresis gel imaging.

[0047...

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Abstract

The invention provides a rapid detection method for p53 gene mutation. The rapid detection method comprises the steps of designing wild type primers and mutant type primers of exon 6 (p53-E6) of p53 and exon 8 (p53-E8) of p53; amplifying a corresponding wild type target segment and mutant type target segment respectively; mixing the above two segments according to different proportions so as to make the content of the mutant type target segments being 1 / 10, 1 / 100, 1 / 1,000 and 0; and finally distinguishing by an HRM method. Compared with other methods, the method provided by the invention has the advantages of high specificity and high sensitivity, is convenient and efficient, and has higher throughput and lower cost for single detection.

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 P53 gene is located on human chromosome 17p13.1, with a total length of about 20Kb, consisting of 11 exons and 10 introns, of which the first exon does not code, and exons 2, 4, 5, 7 and 8 respectively encode five evolutionarily highly conserved domains. The P53 gene encodes a protein consisting of 393 amino acid residues with a molecular weight of 53KD, which is why it is named "P53". [0003] Wild-type P53 protein (wtP53) has the function of regulating cell growth, and multiple pathways inhibit the occurrence of tumors: [0004] ① "The cell cycle arrest P53 protein causes cell G1 phase arrest by regulating the protein encoded by the downstream gene P21; Downstream genes, cell cycle arrest in G2 / M phase. [0005] ② "Promote cell apoptosis; P53 protein can...

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Application Information

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Patent Type & Authority Applications(China)
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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