Specific primer and liquid phase chip for BRAF genetic mutation detection

Abstract

The invention discloses a liquid phase chip for BRAF genetic mutation detection. The liquid phase chip mainly comprises (A) respectively designed ASPE primer pairs in the wild type and in the mutant type directed to a V600E mutation site of the BRAF: each ASPE primer is composed of a tag sequence at a 5'terminus and a specific primer which is at a 3'terminus and directed to a target gene mutation site, one base of last three bases at the 3'terminus of the specific primer is the mutation site, and the Tm value of the specific primer is from 52 to 58 DEG C; (B) two types of microspheres which have color-different codes and are coated with specific anti-tag sequences which can correspondingly have complementary pairing with the selected tag sequence in the (A); and an amplimer for amplifying the target sequence of the BRAF gene having the V600E mutation site. The liquid phase chip for BRAF genetic mutation detection prepared in the invention has an excellent signal to noise ratio, and a cross reaction does not substantially exist between a designed probe and the anti-tag sequence.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to specific primers for BRAF gene mutation detection and a liquid phase chip. technical background [0002] Murine sarcoma viral oncogene homolog B1 (v-taf moourine sarcoma viral oncogene homolog B1, BRAF) gene is located on human chromosome 7q34, and its functional coding region consists of 2510 base pairs, encoding the MAPK pathway The serine-threonine-protein kinase in the cell transduces signals from Ras to MEK1 / 2, thereby participating in the regulation of various biological events in cells. Studies have shown that BRAF mainly has two types of mutations: ① 11% of the mutations are located in the glycine loop on exon 11, such as G463, G465, G468 and other point mutations; ② 89% of the mutations occur in the activation region of exon 15, About 92% of them are located at the 1799th nucleotide (T is mutated to A), resulting in the substitutio...

AI Technical Summary

Problems solved by technology

The PCR-sequencing method has the advantage of being able to determine the range and type of mutations, and is currently a widely used detection method, but the sensitivity of the sequencing method is only 20%-25%, which is far from meeting the needs of practical applications, especially for heterogeneity of tumor somatic mutations, low sensitivity will lead to a large number of missed detections

At the same time, the detection operation of the sequencing method is complicated and the timeliness is poor. For the detection of practical applications that require high timeliness and high sensitivity, the limitations of the sequencing method have long been highlighted

Real-time fluorescence quantitative PCR detection technology has high detection efficiency and strong timeliness, but its high false positive rate is also criticized by practical applications.

Based on the problems of the above-mentioned detection technology, the "BRAF gene mutation detection probe, liquid-phase chip and its detection method" (application number: 200910037357.6) successfully developed by the applicant in the early stage is easy to operate and eliminates a large number of The interference caused by the wild-type sequence has good specificity, high sensitivity, and an accuracy of more than 99%. However, this method involves two rounds of PCR operations, which is more likely to cause pollution, and the probes in the hybridization detection step are relatively close (only the mutation site is different) ), it is not convenient for parallel detection of multiple gene mutation sites

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