Method for detecting methylation level of synuclein intron 1

Abstract

The invention relates to a method for detecting the methylation level of a synuclein intron 1 and belongs to the field of bioengineering. The method comprises the following detection steps of: performing sodium hydrogen sulfite treatment; performing polymerase chain reaction (PCR) amplification; preparing a single chain; preparing a pyrophosphoric acid sequencing sample; preparing before pyrophosphoric acid sequencing analysis; analyzing and calculating the methylation state of a corresponding locus by using computer software. The deoxyribonucleic acid (DNA) methylation detection method provided by the invention overcomes the defects in the existing DNA methylation detection technology and has the advantages of quickness, accuracy, high flux and quantitative property. A method for detecting the CpG island methylation level of an alpha-Syn intron 1 based on a pyrophosphoric acid sequencing platform is established. Electrophoresis, Sanger sequencing and pyrophosphoric acid sequencing prove the detection specificity of the method disclosed by the invention. Compared with a sulfite clone sequencing method, the pyrophosphoric acid sequencing method disclosed by the invention is proved to have a high quantitative property. In addition, the method is convenient and quick to operate and high in flux. 96 samples can be sequenced simultaneously, and the time consumption is less than 2 hours, so the method is extremely suitable for clinical examination work.

Description

technical field [0001] The invention belongs to the field of biological engineering. Background technique [0002] Currently, sulfite sequencing, methylation-specific PCR, and next-generation sequencing are mainly used to detect the methylation level of α-Syn intron 1. Among them, the sulfite sequencing method involves a large amount of amplified fragment cloning work, which is time-consuming and labor-intensive. Also, the ratio of methylated / unmethylated products may shift during cloning, resulting in a loss of quantitative accuracy. The methylation-specific PCR method is relatively simple, but this method can only be used qualitatively, not quantitatively. The next-generation sequencing method is expensive and not suitable for large-scale applications. Contents of the invention [0003] The purpose of the present invention is to provide a detection method capable of quantitatively analyzing the methylation level of CpG island in α-Syn intron 1. [0004] Detection ste...

AI Technical Summary

Problems solved by technology

Among them, the sulfite sequencing method involves a large number of amplified fragment cloning work, which is time-consuming and laborious.

Also, the ratio of methylated / unmethylated products may shift during the cloning process, resulting in a loss of quantitative accuracy

The methylation-specific PCR method is relatively simple, but this method can only be used qualitatively, not quantitatively

The next-generation sequencing method is expensive and not suitable for large-scale applications

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