DNA methylation detection method of specific gene segment

A specific gene and detection method technology, applied in the field of DNA methylation level detection, can solve problems such as difficult to apply to clinical routine detection applications, CpG site methylation, complex data processing, etc., to reduce detection costs and have good applicability , the effect of high sensitivity

Inactive Publication Date: 2016-03-02
GUANGDONG MEDICAL UNIV
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Problems solved by technology

However, there are obvious deficiencies: ①If bisulfite does not treat DNA completely, it will easily lead to false positives; ②The MSP method is designed on the premise that all CpG site cytosines in the primers are completely methylated or completely unmethylated. , and in fact the CpG sites of CpG islands may be partially methylated
However, they are all semi-quantitative methods, and the data processing is complicated, so it is difficult to apply to clinical routine detection applications

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  • DNA methylation detection method of specific gene segment

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Embodiment 1

[0032] Example 1: This method was used to measure the methylation level in exon 2 of the p16 gene in tumor tissue and paracancerous tissue. Note: In this example, the chemical cleavage method is used to cleavage the gene fragment into a single base.

[0033] 1. DNA extraction and bisulfite treatment

[0034] The total genomic DNA of the biological samples was extracted using a commercial DNA extraction kit, and the purity and concentration of the DNA solution were detected by agarose gel electrophoresis and UV-visible spectrophotometry.

[0035]Take a certain amount of genomic DNA, and use a commercial methylation conversion kit for bisulfite treatment to convert unmethylated cytosine (C) into uracil (U), while methylated cytosine constant.

[0036] 2. Acquisition of exon 2 fragment of p16 gene:

[0037] Genomic DNA treated with bisulfite was used as a template, and a pair of primers without CpG sites (upstream: 5'-TGGTAGGTTATGATGATGGGTAG-3', downstream: 5'-CTCAAATCATCAATCC...

Embodiment 2

[0057] Example 2: This method is used to measure the methylation level in exon 2 of p16 gene in tumor tissue. Note: In this example, the enzymatic hydrolysis method was used to cleavage the gene fragments into single nucleosides.

[0058] While keeping the steps of DNA extraction, sulfite treatment, and PCR amplification of gene fragments unchanged, we performed enzymatic hydrolysis on the exon 2 fragment product of p16 gene to obtain single nucleosides, and then used the preferred liquid phase The tandem mass spectrometry program detects the content of the above-mentioned nucleosides, and determines the DNA methylation level of the gene fragment through a formula.

[0059] (1) Cleavage of DNA gene fragments: Denature the gene fragments at 95°C for 5 minutes, and cool on ice for 2 minutes; then add S1 endonuclease and its buffer, and incubate at 37°C for 16 hours; then add alkaline phosphoric acid Salt and its buffer, continue to incubate at 37°C for 4h. The nucleosides gene...

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Abstract

The invention discloses a DNA methylation detection method of a specific gene segment. The method is characterized in that genome DNA of a biological sample is extracted through a commercial kit method, and the specific gene segment is obtained by using the polymerase chain reaction technology after the genome DNA is subjected to bisulfite conversion; a chemical splitting method or enzymolysis method is used to process the target gene segment product to allow the same to be split from a chain sequence into single basic groups or nucleosides; an internal standard method is used to detect the contents of cytosine and adenine or the nucleosides of the adenine through liquid-phase tandem mass spectrum (LC-MS/MS), and the methylation rate of the target gene segment is calculated according to a formula. By the method, the DNA methylation level of the specific gene segment can be detected fast, efficiently and accurately in a quantified manner.

Description

technical field [0001] The invention relates to a method for detecting the DNA methylation level of a specific gene segment in the whole genome of a biological (human, animal, etc.) sample. Background technique [0002] Quantitative analysis of DNA methylation of specific genes is of great significance to cancer research and diagnosis. Bisulfite sequencing PCR (BSP) combined with bisulfite treatment is recognized as the gold standard for methylation analysis. The basic process of this method is: after the DNA is treated with bisulfite, the unmethylated cytosine is deaminated and converted into uracil, while the methylated cytosine remains unchanged. After PCR amplification, the methylated cytosine Cytosine is amplified to guanine and uracil is amplified to thymine. The PCR product is cloned and then sequenced. Through sequence comparison with the gene bank and statistical analysis of the data, it is possible to determine whether the CpG site is methylated and the degree of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858
Inventor 蔡春叶晓霞张立坚黄琼林张俊杰陈光照
Owner GUANGDONG MEDICAL UNIV
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