Method for detecting tumor mutant gene in blood

Abstract

The invention relates to a method for detecting a tumor mutant gene in blood. The method comprises the following steps of: (1) preparing a polyethylene oxide sieving medium containing TBE (Tetrabromoethane); (2) extracting a genome DNA (Deoxyribonucleic Acid) in a blood sample; (3) amplifying a gene target fragment needing to be detected: performing conventional PCR (Polymerase Chain Reaction) amplification by taking the genome DNA as an amplifying template to obtain a p53 gene PCR amplification sample and a k-ras gene PCR amplification sample; (4) treating the samples: performing denaturation or enzyme digestion on the p53 gene PCR amplification sample and the k-ras gene PCR amplification sample to obtain a denatured sample solution and an enzyme digestion solution respectively; and (5) detecting the samples: adding a 10*SYBR Green I fluorescence dye and the genome DNA into the denatured sample solution and the enzyme digestion solution respectively, uniformly mixing, adding deionized water till a system is 30-50 mu l, automatically filling the polyethylene oxide sieving medium by using a capillary electrophoresis apparatus pressure system and performing CE-SSCP (Capillary Electrophoresis-Single Strand Conformation Polymorphism) detection or CE-RFLP (Capillary Electrophoresis-Restriction Fragment Length Polymorphism) detection respectively. The method is easy to operate, and has high repeatability.

Description

technical field [0001] The invention relates to a capillary electrophoresis detection method in the field of analysis method application research, in particular to a method for detecting tumor mutation genes in blood. Background technique [0002] The detection of gene mutation plays a very important role in clinical disease diagnosis. At present, PCR-based polyacrylamide gel (PAGE) electrophoresis and various gene mutation detection techniques are often used to screen gene point mutations. These methods have certain advantages in meeting the needs of clinical rapid detection of gene mutations. limitation. Therefore, it is imperative to establish an accurate and rapid gene mutation detection method for clinical diagnosis. [0003] Capillary electrophoresis (CE) is a new separation and analysis technology combining electrophoresis and chromatography. It has the characteristics of high separation efficiency, fast analysis speed, less sample consumption and wide application r...

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Problems solved by technology

In the Chinese patent database from 1984 to 2010, the Chinese patent with the application number 200510026931.X "A PCR detection method and reagent system for tumor-related gene mutations" and the Chinese patent with the patent number 200910042599.4 "for the individualization of malignant tumors Gene chip and its application for drug-related gene mutation detection", Chinese patent "Method for detecting gene mutation" with application number 200810005523.X, and Chinese patent "Rapid detection of APC gene mutation" with application number 201010134207.X 200910201917.7 Chinese patent "A Primer for Detecting K-ras Gene Mutation and Its Application", the above-mentioned patent only discloses gene mutations using real-time quantitative fluorescent PCR, sequencing, high-resolution melting curve method, fluorescent double-loop probe technology, etc. The detection method detects tumor gene mutations in tissue or blood, but the method and application of capillary electrophoresis combined with SSCP and RFLP for detecting tumor mutation genes in blood have not been reported in the literature

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