A method for quantitative detection of methylation by pyrosequencing

Abstract

The invention discloses a method for quantitative determination of methylation through pyrosequencing. According to the method, PCR products of the genome DNA obtained through conversion of the hydrosulphite rapidly pass through areas 'A', 'G' and 'T' in the DNA template sequence according to two specific nucleotides, while dGTP is added into a measurement DNA template sequence for being subjected to pyrosequencing through a 'C' content method, and each sequencing reaction is continuously recorded to obtain information, namely, the encoding information of synthesis number of the two nucleotides and the information of the synthesis number of dGTP are obtained, through the setting that basic groups C in all the GC sites in the determined object region sequence are converted into T at different degrees, then the information of the synthesis number of dGTP is utilized, so that the quantitative analysis on the methylation sites in the object region sequence is realized. According to the method provided by the invention, the sequencing length can be greatly improved, the analysis range of the traditional pyrosequencing is expanded, and the method is suitable for methylation quantitative analysis for sequences of single-sample PCR products and mixed-sample PCR products.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a PCR product sequence analysis method, in particular to a method for quantitatively detecting methylation by synthetic pyrosequencing with specific nucleotide addition. Background technique [0002] The development and completion of the Human Genome Project and various model organism genome projects has brought mankind into the post-gene era, which has had a huge impact on contemporary biological and medical research, and the related disciplines of molecular biology have developed rapidly. It will become possible to understand the differences of life, the law of disease occurrence and development, and the interaction between drugs and living organisms at the genetic level. As far as gene sequence analysis is concerned, the focus of the post-genome era has shifted from whole-genome sequence determination to the comparison of individual genetic differences and inter-species genetic diff...

AI Technical Summary

Problems solved by technology

However, on the one hand, the use of multiple sequencing primers will also increase the analysis cost; on the other hand, since the methylated cytosine remains unchanged after bisulfite treatment, the methylated cytosine at each site In different degrees, the content of the two DNA templates in the PCR product is also different, resulting in complex types of DNA templates, and it is generally difficult to design multiple sequencing primers to segment their sequences for determination

Therefore, traditional pyrosequencing is insufficient in the quantitative detection of methylation in long fragment sequences

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