A method for accurately verifying the degree of methylation of unknown genes

Abstract

The invention relates to a method capable of accurately verifying the methylation degree of unknown genes. The method is a latest method capable of accurately verifying methylation degree of unknown genes. The latest method comprises the following steps: respectively setting specific primers of target genes of an experiment group and a control group, adding a transforming agent into a product by difference qualification of a high-resolution melting (HRM) curve, adding DNA excision enzyme for enzymolysis, accurately quantifying the percentage of the content of cytosine or adenine by LC-MS / MS, summarizing a relation between canceration probability and methylation degree in differentiation of cells or tissues according to qualitative and quantitative data obtained by experiments in two steps to build a relational database so as to come into play in the super early diagnosis of cancer.

Description

technical field [0001] The invention relates to a method for verifying the degree of methylation of a gene, in particular to a method for accurately verifying the degree of methylation of an unknown gene by using high-resolution melting curve (HRM) and LC-MS / MS technology. Background technique [0002] 1. High resolution melting curve (HRM) and LC-MS / MS technology [0003] a) Principle of HRM technology: [0004] High-resolution melting curve analysis (HRM) is to monitor the combination of double-stranded DNA fluorescent dye and PCR amplification product in real time during the heating process. Due to the mismatch of single nucleotide polymorphism (SNP) sites, the double-stranded DNA will be untied first during the heating process, and the fluorescent dye will be released from the partially unzipped DNA molecules, which can be judged from the fluorescence intensity and time curve Whether there is a SNP, and different SNP sites, heterozygotes and homozygotes will affect the...

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Problems solved by technology

However, only obtaining this data cannot fundamentally solve the proportional relationship between high-throughput data and cancer diagnosis

If product sequencing is used, although point mutations or methylation sites can be resolved, firstly, the cost is high and the sequencing time period is long; secondly, because mutations do not exist in all samples, a large amount of data is redundant data, such as nonsense mutations , synonymous mutations, or false-positive mutations caused by sequencing errors. These reasons will increase the cost of sequencing and the difficulty of subsequent data processing. The blindness is greater, which is very unfavorable for high-throughput screening of effective data. [2，3] Once again, the speed and resolution of the traditional HPLC method for quantification are difficult to achieve the degree of accurate quantification. Since the traditional HPLC method is based on the detection of ultraviolet absorbance, it is difficult to detect trace single nucleotides by using the mass-to-charge ratio of the secondary ion. For mass spectrometry changes, the specificity is not strong, and the resolution and precision are not as accurate as mass spectrometry detection.

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