Method and kit for quantitatively detecting methylation degree of DNA specific site

Abstract

The invention provides a method and kit for quantitatively detecting the methylation degree of a DNA specific site. The method provided by the invention comprises the following steps: 1) designing a PCR primer in accordance with a DNA sequence to be detected, and using the PCR primer for amplifying the DNA to be detected; 2) designing a pair of TaqMan probes in accordance with a CpG site-enriched region in the PCR product obtained by amplification in the step 1), wherein one probe is a CG probe designed in accordance with the DNA of which the CpG site is completely methylated, and the other probe is a TG probe designed in accordance with the DNA of which the CpG site is completely non-methylated and is used for specifically combining the PCR product and detecting the PCR product; and 3) carrying out real-time quantitative PCR based on the primer designed in the step 1) and the probes designed in the step 2), thus detecting the methylation degree of the DNA to be detected. By using the method, a specific methylated site can be detected, and the methylation states of other sites can not affect the detection result. The kit provided by the invention can be used for detecting the methylation degree of a DNA specific site.

Description

technical field [0001] The invention relates to a method and a kit for detecting DNA methylation, in particular to a method and a kit for quantitatively detecting the methylation degree of a specific DNA site. Background technique [0002] DNA methylation is an important part of epigenetics, which plays an important role in maintaining normal cell function, genetic imprinting, embryonic development and human tumorigenesis, and is one of the new research hotspots. [0003] DNA methylation is one of the earliest epigenetic modifications discovered, and it exists in the DNA genetics of all organisms. DNA methylation can turn off or turn on the activity of certain genes, while demethylation induces or inactivates the reactivation and expression of genes. The main forms of methylation are 5-methylcytosine, N6-methyladenine and 7-methylguanine. CCA / TGG and GATC are often methylated in prokaryotes, while methylation only occurs at cytosine in eukaryotes. DNA methylation is the c...

AI Technical Summary

Problems solved by technology

However, in the actual clinical application process, due to the difficulty in obtaining uniform clinical samples with complete lesions, it is inevitable to incorporate methylation or non-methylation factors of normal samples into the lesion samples during the above real-time PCR detection process. , resulting in an error in the determination of the degree of methylation, thus affecting the accuracy of the test results

In this case, the above-mentioned real-time PCR method for detecting methylation at a specific site is limited.

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