Method and kit for detecting mutation of K-ras gene

Abstract

The invention discloses a method and kit for detecting the mutation of a K-ras gene by using PCR (Polymerase Chain Reaction) amplification, belonging to the field of molecular biology. The kit comprises a conventional PCR assembly and a specific primer, and the specific primer comprises three forward primers and one reverse primer, wherein the sequences of the three forward primers respectively have nucleotide sequences represented by SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3; and the reverse primer has a nucleotide sequence represented by SEQ ID No.4. The existence of a target product band of PCR amplification through electrophoretic separation is used for judging whether the K-ras gene has at least one of three hot spot mutations including GGT-GAT and GGT-GTT of No.12 codon and GGC-GAC of No.13 codon. The kit and the method for detecting the mutation of a K-ras gene provided by the invention have the advantages of quickness, accuracy, simple method and low cost, in favor of clinic application and popularization.

Description

Technical field [0001] The invention belongs to the field of molecular biology, and relates to a method and a kit for detecting K-ras gene mutations by PCR amplification. Background technique [0002] K-ras gene is an important proto-oncogene. The mutation of K-ras gene keeps the ras protein in an activated state, which stimulates the continuous growth or differentiation of cells, and ultimately leads to malignant transformation of cells. K-ras gene mutations occur in approximately 30% of human tumor cells. Bosetal analyzed the incidence of K-ras gene point mutations in human tumors. Pancreatic cancer 82%, colon cancer 43%, lung cancer 30%c, thyroid cancer 29%, bladder, liver, kidney and uterine cancer 10% or less %. Mok et al. reported that the incidence of K-ras mutations in borderline tumors was 48%, and the mutations occurred in codon 12 and codon 13, respectively. The point mutation rate of K-ras gene in colorectal cancer is more than 40%-50%, and the point mutations are ...

AI Technical Summary

Problems solved by technology

In June 2008, the latest clinical research results were released at the American Society of Clinical Oncology (ASCO) annual meeting, that is, patients with K-ras mutations do not benefit from anti-EGFR therapy, but instead increase the risk of adverse reactions and treatment costs; and K-ras wild-type patients are likely to benefit from this type of drug treatment

The false negatives obtained by the above different methods are different, but the common low-fidelity polymerase without correction function makes all these methods have their structural defects, that is, they inevitably produce high false positives

However, the currently routinely used Sanger enzymatic sequencing, that is, single-base extension reaction mediated by dideoxynucleotides, can detect azyme with low mutation content, mitochondrial heteroplasm with few mutations, somatic mutation and frequency distribution. Low DNA pool analysis are difficult to apply

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