Few-sample whole genome DNA (deoxyribonucleic acid) methylation detection method and kit

Abstract

The invention discloses a few-sample whole genome DNA (deoxyribonucleic acid) methylation detection method, and relates to a method for detecting less-sample (300pg DNA or 50 cells) whole genome DNA methylation by an improved MeDIP-Seq technique based on high-throughput sequencing. According to the method, lambda DNA containing dUTP is added into a small number of initial samples to reduce sampleloss, base-building and immuno-precipitation efficiency is improved, MeDIP-DNA is digested by USER enzyme, so that added methylation lambda DNA is removed, and a methylation sample sequencing libraryto be detected cannot be polluted by the methylation lambda DNA. According to the method, needed sample amount is 300pg DNA or 50 cells, the method is more applicable to MeDIP-Seq analysis of a smallnumber of source samples, operation method of a MeDIP experimental technique and a kit matched with the experimental technique is simple and convenient, specific commercial kits and experimental devices are omitted, cost is low, and the method has wide applicability and universality.

Description

technical field [0001] The present invention relates to a method for detecting DNA methylation in the whole genome, in particular to a method for detecting whole genome DNA in a small amount of samples (300pg DNA or 50 cells) by improving MeDIP-Seq (Methylated DNA immunoprecipitation sequencing) technology based on high-throughput sequencing method of methylation. Background technique [0002] At present, the minimum amount of DNA required to detect the DNA methylation of the whole genome of cells by MeDIP-seq method is 50ng. If cells are used as the starting sample, at least ~75000 cells are required (Taiwo O et al., Nat Protoc, 2012), The specific experimental process of this method is: extraction of cellular genomic DNA → ultrasonic fragmentation of genomic DNA and detection of ultrasonic fragment size by Agilent Bioanalyzer 2100 → end repair of ultrasonic fragments, addition of dA at the 3' end, adapter ligation, and purification and quantification of the ligated product...

AI Technical Summary

Problems solved by technology

[0004] There are two main disadvantages of the existing technology: ①The minimum required sample input amount is 50ng DNA or 75,000 cells, which is still not suitable for MeDIP-seq analysis of a small amount of source samples; ②It needs to use commercial kits and fully automatic Sample processing system for MeDIP-seq experiments is expensive and not universally applicable

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