The bisulfite genomic sequencing protocol

Jane J Pappas,Walter Edward Clarke Bradley,André Toulouse

doi:10.4236/alc.2013.21004

Jane J Pappas, Walter Edward Clarke Bradley + Show 1 more

Open Access

https://doi.org/10.4236/alc.2013.21004

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Abstract

The bisulfite genomic sequencing (BGS) protocol has gained worldwide popularity as the method of choice for analyzing DNA methylation. It is this popular because it is a powerful protocol and it may be coupled with many other applications. However, users often run into a slew ofproblems, including incomplete conversion,overly degraded DNA, sub-optimal PCR amplifications, false positives, uninformative results, or altogether failed experiments. We pinpoint the reasons why these problems arise and carefully explain the critical steps toward accomplishing a successful experiment step-by-step. This protocol has worked successfully (>99.9% conversion) on as little as 100 ng of DNA derived from nearly 10-year-old DNA samples extracted from whole blood stored at -80°C and resulted in enough converted DNA for more than 50 PCRreactions. The aim of this article is to makelearning and usage of BGS easier, more efficient and standardized for all users.

Highlights

The bisulfite genomic sequencing protocol (BGS) has gained worldwide popularity as the method of choice to analyze DNA methylation
DNA methylation was the first epigenetic mark to be discovered in mammalian cells [1], and while many other epigenetic marks are known and even more are currently being discovered [2], the role that DNA methylation plays in the regulation of gene expression is widely accepted
Conversion can only occur in single-stranded DNA

Summary

Introduction

The bisulfite genomic sequencing protocol (BGS) has gained worldwide popularity as the method of choice to analyze DNA methylation. Bisulfite was first used to convert 5-methylcytosines to uracils in 1970 [3], but the BGS protocol used today to determine the methylation status of CpG dinucleotides in the genome was first published in 1992 [4]. Sodium bisulfite and metabisulfite ions are used to convert unmethylated cytosines to uracils in three steps (Figure 1). Bisulfite does not affect 5-methylcytosines: the methyl group in the 5 position of the cytosine base causes 5-methylcytosines to be non-reactive to bisulfite ions. The end result following PCR amplification of bisulfite-converted DNA is that 5-methylcytosines in the original sample are read as C, whereas cytosines are read as T (Figure 2)

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Discussion

Conclusion