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Bisulphite differential denaturation PCR for analysis of DNA methylation

Abstract

Differential denaturation during PCR can be used to selectively amplify unmethylated DNA from a methylated DNA background. The use of differential denaturation in PCR is particularly suited to amplification of undermethylated sequences following treatment with bisulphite, since bisulphite selectively converts cytosines to uracil while methylated cytosines remain unreactive. Thus amplicons derived from unmethylated DNA retain fewer cytosines and their lower G + C content allows for their amplification at the lower melting temperatures, while limiting amplification of the corresponding methylated amplicons (Bisulphite Differential Denaturation PCR, BDD-PCR). Selective amplification of unmethylated DNA of four human genomic regions from three genes, GSTP1, BRCA1 and MAGE-A1, is demonstrated with selectivity observed at a ratio of down to one unmethylated molecule in 10(5) methylated molecules. BDD-PCR has the potential to be used to selectively amplify and detect aberrantly demethylated genes, such as oncogenes, in cancers. Additionally BDD-PCR can be effectively utilized in improving the specificity of methylation specific PCR (MSP) by limiting amplification of DNA that is not fully converted, thus preventing misinterpretation of the methylation versus non-conversion.

Type Journal
ISBN 1559-2308 (Electronic)
Authors Rand, K. N.;Mitchell, S. M.;Clark, S. J.;Molloy, P. L. :
Garvan Authors Prof Susan Clark
Publisher Name Epigenetics
Published Date 2006-01-01 00:00:00
Published Volume 1
Published Issue 2
Published Pages 94-100
URL http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17998815
Status Published In-print
OpenAccess Link https://publications.gimr.garvan.org.au/download.php?2116_10600/06 Rand EPI-06.pdf