PCR primer group, kit and application thereof

Abstract

The invention discloses a PCR primer group, a kit and application thereof. The total number of the PCR primer group is 65. The primer pairs are uniformly distributed in the full length of the RB1 gene, and meanwhile, extra densification is carried out on a mutation hot spot region. The primer pair of the invention can be used for full-length amplification of exons which are short in part of the RB1 gene and are close to each other, and deletion in the amplification region can be found. For an intron region with a relatively long span, a primer pair design is additionally added, aiming at a relatively conserved sequence of an intron, a large-section repetitive sequence and a high-GC-ratio sequence are avoided, and a PCR reaction is easy to carry out. By using the method, the breakpoint of RB1 gene exon deletion can be simply and conveniently positioned with extremely low price, basic molecular biology experiment conditions and relatively low labor hours. The PCR primer group can be used for primary screening of RB1 gene exon deletion of small-scale tumor samples, and can also be used for accurately positioning breakpoints of RB1 gene exon deletion in combination with WES results.

Description

technical field [0001] The invention relates to the field of medical diagnosis, in particular to a PCR primer set, a kit comprising the primer set and the application of the primer set or the kit in locating the RB1 gene exon deletion breakpoint. Background technique [0002] RB1 gene is the first tumor suppressor gene discovered by humans, which plays an important role in regulating cell cycle and inhibiting cell proliferation. Inactivating mutations in the RB1 gene can lead to uncontrolled proliferation of tumor cells and are an important driver mutation in malignant tumors. Germline mutations in the RB1 gene can lead to retinoblastoma. Sequencing studies on human malignant tumors have confirmed that the mutation frequency of RB1 gene in different solid tumors is 10%-30%. In some tumors, especially small cell lung cancer, the mutation frequency of the RB1 gene is as high as 90%. [0003] Recent publications have shown that there are exon deletions of the RB1 gene in tum...

AI Technical Summary

Problems solved by technology

The exon deletion length of the RB1 gene is much smaller than the mega base (Mb) deletions that can be detected by traditional sequencing studies or single nucleotide polymorphism (SNP) chips, using traditional sequencing method difficult to detect

[0005] (1) SNP chips can detect gene deletions up to several Mb long, but the exon deletion length of RB1 gene is only tens of kb to hundreds of kb, which cannot be detected by SNP chip

[0006] (2) The currently commonly used whole-exome sequencing (whole-exome sequencing, WES) method only captures and sequences the exons of the RB1 gene, and it can be speculated that some RB1 gene exon deletion events, but may miss some RB1 gene exons. Exon deletions; and inability to pinpoint deletion breakpoints located in introns

[0007] (3) Although whole-genome sequencing (whole-genome sequencing) sequencing or third-generation long-read sequencing (longread sequencing) technology can locate and delete breakpoints more accurately, it is expensive, requires large-scale special equipment, and is not easy to carry out on a large scale

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