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PCR-RFLP method for detecting H-site mutation of PIK3CA gene

A PCR-RFLP, site mutation technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., to achieve the effects of good detection reproducibility and accuracy, improved accuracy, and improved sensitivity

Inactive Publication Date: 2015-05-06
中国医科大学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Compared with direct DNA sequencing, this method has higher detection sensitivity; compared with HRM and other methods that use peak shape differences to judge results, this method has higher detection accuracy, and has no complicated equipment requirements, and the detection cost is relatively low , suitable for practical clinical application, but there is no report on the detection of PIK3CA gene mutation using this method

Method used

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  • PCR-RFLP method for detecting H-site mutation of PIK3CA gene
  • PCR-RFLP method for detecting H-site mutation of PIK3CA gene

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Embodiment 1

[0047] The PCR-RFLP method of the present invention detects the H1047R site mutation of the PIK3CA gene in colorectal cancer cells comprising the following steps:

[0048] (1) Genomic DNA extraction from samples

[0049] Human colorectal cancer cell lines SW620 and LS174T in the logarithmic growth phase were respectively taken, digested with trypsin, and counted. Take cells 1 x 10 6centrifuge at 1000 rpm for 5 min, discard the supernatant; extract the genomic DNA of the cells according to the operation steps of the Promega Whole Genome Extraction Kit; dissolve the extracted DNA in 20 μL ddH 2 O, measured at a wavelength of 260 nm and adjusted to a concentration of 0.5 μg / μL;

[0050] (2) PCR amplification of PIK3CA gene

[0051] Using about 100 ng of extracted genomic DNA as a template, PCR amplification was performed using the upstream primer 5'-GGAGTATTTCATGAAACAAATGAATGATGCG-3' and the downstream primer 5'-GAGCTTTCATTTTCTCAGTTATCTT-3' to obtain a 126bp amplified product;...

Embodiment 2

[0074] The PCR-RFLP method of the present invention detects the H1047R site mutation of the PIK3CA gene in the paraffin section specimen of the tumor tissue of a patient with colorectal cancer, comprising the following steps:

[0075] (1) Genomic DNA extraction from samples

[0076] Take paraffin sections (5 μm thickness) of tumor tissues from patients with colorectal cancer, extract the genomic DNA of the cells according to the operation steps of the OMEGA Paraffin Section DNA Extraction Kit, and finally elute the DNA with 40 μL of eluent for later use;

[0077] (2) PCR amplification of PIK3CA gene

[0078] Take 2 μL of the DNA eluate as a template, use the upstream primer 5'-GGAGTATTTCATGAAACAA ATGAATGATGCG-3' and the downstream primer 5'-GAGCTTTCATTTTCTCAGTTATCTT-3' to perform PCR amplification to obtain a 126bp amplification product;

[0079] The total volume of the PCR reaction is 25 μL, and the composition is:

[0080] wxya 2 O 16 μL

[0081] 10×buffer (with Mg 2+ )...

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Abstract

The invention discloses a PCR-RFLP method for detecting H1047R-site mutation of a PIK3CA gene. According to the method, a pair of specific primers are designed for the H1047R mutation site of the twentieth codon of the PIK3CA gene; and an Fst I digestion site is introduced to establish the PCR-RFLP method. The PCR-RFLP method mainly comprises the following steps: (1) extracting the genome DNA of a sample to be detected; (2) carrying out PCR amplification on a PIK3CA gene segment which contains the H1047R mutation site; (3) taking the PIK3CA gene segment as a template to carry out Fst I restriction enzyme reaction; (4) carrying out gel electrophoresis to obtain a length polymorphism atlas of restriction segments; and (5) determining the PIK3CA gene mutation state of the sample to be detected according to the length and quantity of the restriction fragments. The method has the advantages of being simple to operate, strong in specificity, high in sensitivity and low in cost, and can be used for effectively detecting the PIK3CA gene mutation state.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a PCR-RFLP method for detecting the H1047R site mutation of PIK3CA gene. Background technique [0002] Phosphatidylinositol 3-kinase (PI3K) is an enzyme that phosphorylates the 3-hydroxyl of the inositol ring in the phosphatidylinositol molecule. The signaling pathways regulated by it involve cell proliferation, adhesion, survival and other life activities. Play an important role in the development of tumors. The PIK3CA gene encodes the catalytic subunit of PI3K, which can activate the downstream AKT pathway of the PI3K pathway. The mutation of PIK3CA is closely related to the occurrence of various tumors such as colorectal cancer, breast cancer, lung cancer and ovarian cancer. Studies have found that in colorectal cancer, the mutation hotspots of PIK3CA are concentrated in E542K, E545K and H1047R. Recent studies have shown that the H1047R point mutation located in exo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2521/301C12Q2531/113
Inventor 方瑾李婉明周婷婷冯一鸣
Owner 中国医科大学
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