Septin 9 gene methylation detection method and kit

Abstract

The invention provides a Septin 9 gene methylation detection kit. The kit comprises at least one of the following three groups of specific primers and probes aiming at the detection of Septin 9-v2 exon methylation, SEQ ID NO.5-SEQ ID NO.7, SEQ ID NO.8-SEQ ID NO.10, and SEQ ID NO.11-SEQ ID.13. The invention further provides a Septin 9 gene methylation detection method and a sample pretreatment method. The Septin 9 gene methylation detection method comprises the following steps of TET enzyme oxidation; pyridine borane reaction; and multiple fluorescence PCR reaction. The kit and the detection method have the advantages of high detection sensitivity, good specificity and high detection flux.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method and a kit for detection of human gene methylation. Background technique [0002] The Septin9 gene is one of the members of the Septin gene family discovered by Hartwell et al., and has guanosine triphosphatase (GTPase) activity. The Septin9 gene is located on human chromosome 17q25.3, contains 17 exons, and is about 2.40×10 5 bp, encoding Septin 9 protein. However, because its transcripts contain multiple translation origins and variant splicing, Septin9 can mutate to produce 18 variant splicing forms and encode 15 polypeptides. The subtypes of Septin9 have 75% nucleotide sequence similarity and 89%-96% amino acid identity. In addition, the distribution of these variant splicing bodies is tissue-specific. For example, the variant splicing body Septin 9-v1 of the Septin9 gene is distributed in tissues other than the brain and thymus, and the variant splicing bod...

AI Technical Summary

Problems solved by technology

However, the kit only detects the methylation status of a region of the Septin 9 gene, and the popularity of its applicable detection models is low, which limits its clinical promotion and application

[0007] In addition, it is worth noting that although the bisulfite conversion method is considered to be the "gold standard" for DNA methylation analysis, it has two major disadvantages: 1) The conditions of bisulfite conversion are too severe, and DNA may 2) Bisulfite conversion completely converts unmodified cytosine into thymine (unmodified cytosine accounts for about 95% of the total cytosine in the human genome), converting all The conversion of cytosine to thymine severely reduces sequence complexity, leading to a series of problems such as poor detection quality, low mapping rate, uneven genome coverage and increased detection cost

Therefore, the inherent defects of the bisulfite conversion method, the single problem of the Septin 9 gene fragment for methylation detection, and the problem of applicable models are problems that must be solved in the current Septin 9 gene methylation detection

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