Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA

Author: Warnecke Peter M.   Stirzaker Clare   Melki John R.   Millar Douglas S.   Paul Cheryl L.   Clark Susan J.  

Publisher: Oxford University Press

ISSN: 1362-4962

Source: Nucleic Acids Research, Vol.25, Iss.21, 1997-11, pp. : 4422-4426

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Abstract

Methylation analysis of individual cytosines in genomic DNA can be determined quantitatively by bisulphite treatment and PCR amplification of the target DNA sequence, followed by restriction enzyme digestion or sequencing. Methylated and unmethylated molecules, however, have different sequences after bisulphite conversion. For some sequences this can result in bias during the PCR amplification leading to an inaccurate estimate of methylation. PCR bias is sequence dependent and often strand-specific. This study presents a simple method for detection and measurement of PCR bias for any set of primers, and investigates parameters for overcoming PCR bias.