Kit and method for rapidly detecting DNA methylation

Abstract

The invention discloses a kit and method for rapidly detecting DNA methylation. Compared with traditional method, the method of the invention has the following four improvements: 1) DNA extraction and DNA modification are completed synchronously; 2) vulcanization accelerator MBT and guanidinium isothiocyanate are added in modifying solution so as to rapidly complete the DNA extraction and DNA modification processes; 3) the modification reaction adopts a short-time high-temperature processing method to ensure that the DNA can be in the desmolysis state, basic groups are fully exposed and cytosines which are not modified through methylation are converted to sulphonated uracils; and 4) the complicated dialysis desalination step after modification is simplified, namely the modified product istransferred in a nucleic acid purification column, then elution is performed after the column is processed with cleaning solution; and the DNA template of the methylated polymerase chain reaction (PCR) of the detected gene can be obtained. The method of the invention has the advantages of low cost, time saving and good repeatability and can be directly used to detect the methylation level of various tissue cell genes and has important use value in the biomedical detection technical aspects such as the early clinical diagnosis.

Description

Technical field: [0001] The invention relates to the technical field of biomedical detection, in particular to a kit and method for rapidly detecting DNA methylation. Background technique: [0002] DNA methylation is an important part of epigenetics (Epigenetics), which is the conversion of cytosine at the 5' end of CpG dinucleotides to 5' methylcytosine under the action of DNA methyltransferases (DNMTs) . In mammals, the frequency of CpG sequences in the genome is only 1%, but in some regions of the genome, usually located in the promoter region or the first exon region of the gene, the CpG sequence density is very high, which can More than 5 times the average value, it becomes the enrichment area of ​​guanine and cytosine, forming the so-called CpG island. DNA methylation plays an important role in normal human development, X chromosome inactivation, aging, and many human diseases (such as developmental deformities, cancer, cardiovascular diseases, diabetes, and neuropsy...

AI Technical Summary

Problems solved by technology

[0018] (1) The required amount of DNA is 1-2ug, which requires a large amount of human blood or tissue to be tested in clinical diagnosis, which brings unnecessary pain to the tested person

[0019] (2) The operation is cumbersome, DNA must be extracted first, and then DNA modification is carried out, especially in the bisulfite modification process, which takes 16-18 hours; the purification and desulfo steps are also very long, which seriously affects the work efficiency, and, easy to cause pollution

[0020] (3) Low temperature modification at 50°C for 16-18 hours cannot fully modify, because at this temperature, the supercoiled structure of DNA cannot be opened, resulting in incomplete modification and low repeatability of the experiment

[0021] (4) At present, there are various methylation detection kits on the market. For example, the kit produced by GENMED SCIENTIFICSINC in the United States has 14 kinds of solutions, and the operation steps are as many as 30 steps, which is quite cumbersome; the EZ DNA Methylation- Although the Gold Kit is easy to operate, it is expensive, and the amount of DNA processed each time is only enough to do 2 to 3 PCRs

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