Protocol

Amplification of Bisulfite-Converted DNA for Genome-Wide DNA Methylation Profiling

Department of Plant Biology, University of Geneva, Sciences III, CH-1211 Geneva 4, Switzerland

Corresponding author (Jon.Reinders{at}unige.ch).

INTRODUCTION

DNA methylation, in which cytosine is modified to form 5-methylcytosine, is a well-characterized epigenetic modification essential for normal development in plants and mammals. Aberrant DNA methylation patterns have been implicated in disease development, notably cancer. It is therefore important to develop genome-wide DNA methylation profiling technology. In this article, I describe a bisulfite methylation profiling (BiMP) method for assessing DNA methylation patterns. Treatment of DNA with sodium bisulfite converts unmethylated cytosine to uracil, whereas 5-methylcytosine is not converted. During subsequent polymerase chain reaction (PCR) amplification, uracil amplifies as thymine, whereas 5-methylcytosine amplifies as cytosine, thereby creating C-to-T transitions. Although single base detection of DNA methylation patterns provided by bisulfite conversion is clearly advantageous, there are inherent challenges associated with this approach. Successful conversion requires relatively high temperatures (50°C-60°C) and low pH (5.2), both of which may result in a high degree of DNA fragmentation. Also, whole genome amplification methods established for genomic DNA have resulted in poor reproducibility when applied to bisulfite-converted DNA. The protocol described here mitigates these adverse effects and provides bisulfite-converted DNA suitable for genome-wide DNA methylation profiling using array-based analysis platforms.