B-raf gene V600E mutation detection method

Abstract

The present invention relates to a B-raf gene V600E mutation detection method, and belongs to the field of life science and technology, wherein the method can be used for detecting whether a particular nucleotide variation exists in the B-raf gene in the DNA sample isolated from the patient body in vivo. The method comprises a polymerase chain reaction, wherein the reaction system contains a specific forward primer, a specific downstream primer, and a set of probes treated according to a specific method, a single-stranded nucleotide hybridization and melting process is performed under specific conditions, and a fluorescence quantitative PCR instrument is utilized to collect the fluorescence signal during the melting so as to determine the presence or absence of the variation.

Description

Technical field: [0001] The invention relates to a method for diagnosing the V600E mutation of the B-raf gene. The method can detect whether there is a specific nucleotide variation in the B-raf gene in a DNA sample isolated from a patient. It belongs to the field of life science and technology. Background technique: [0002] The B-raf gene is an oncogene, which belongs to the RAF family with the ARAF and RAF1 (CRAF) genes. It is located at 7q34 and is about 190kb long. The B-raf protein translated by it consists of 783 amino acids and has three conserved regions, CR1, CR2 and CR3. B-raf protein is a member of serine-threonine protein kinase raf / mil family and an important regulator in the Ras-Raf-MEK-ERK signaling pathway. The B-raf protein is located at the entrance of the MAPK / ERK pathway, and it connects receptors and RAS proteins on the cell surface with transcription factors in the nucleus through MEK and ERK, thereby regulating cell differentiation and division. Wh...

AI Technical Summary

Problems solved by technology

This method has the following disadvantages: the sensitivity of detection is not high enough: if the content of the mutated gene accounts for less than 10% of the total amount of genomic DNA, the existence of the mutated sample cannot be detected by direct sequencing; High requirements; non-closed-tube operation involves the operation after PCR amplification, so it is easy to be contaminated, resulting in unsatisfactory results; the interpretation of sequencing results is highly subjective; the number of samples tested in one experiment is limited, at most 8-24 example

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