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DNA methylation: bisulphite modification and analysis

Abstract

DNA methylation is an important epigenetic modification of DNA in mammalian genomes. DNA methylation patterns are established early in development, modulated during tissue-specific differentiation and disrupted in many disease states, including cancer. To understand further the biological functions of these changes, accurate and reproducible methods are required to fully analyze the DNA methylation sequence. Here, we describe the 'gold-standard' bisulphite conversion protocol that can be used to re-sequence DNA from mammalian cells in order to determine and quantify the methylation state of a gene or genomic region at single-nucleotide resolution. The process of bisulphite treatment exploits the different sensitivities of cytosine and 5-methylcytosine (5-MeC) to deamination by bisulphite under acidic conditions--in which cytosine undergoes conversion to uracil, whereas 5-MeC remains unreactive. Bisulphite conversion of DNA, in either single tubes or in a 96-well format, can be performed in a minimum of 8 h and a maximum of 18 h, depending on the amount and quality of starting DNA.

Type Journal
ISBN 1750-2799 (Electronic)
Authors Clark, S. J.;Statham, A.;Stirzaker, C.;Molloy, P. L.;Frommer, M. :
Responsible Garvan Author Prof Susan Clark
Publisher Name Nature Protocols
Published Date 2006-01-01
Published Volume 1
Published Issue 5
Published Pages 2353-64
Status Published in-print
URL link to publisher's version http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17406479
OpenAccess link to author's accepted manuscript version https://publications.gimr.garvan.org.au/open-access/2040