DETECTION AND MEASUREMENT OF PCR BIAS IN QUANTITATIVE METHYLATION ANALYSIS OF BISULFITE-TREATED DNA

Methylation analysis of individual cytosines in genomic DNA can be det ermined quantitatively by bisulphite treatment and PCR amplification o f the target DNA sequence, followed by restriction enzyme digestion or sequencing. Methylated and unmethylated molecules, however, have diff erent sequences after bisulphite conversion. For some sequences this c an result in bias during the PCR amplification leading to an inaccurat e estimate of methylation. PCR bias is sequence dependent and often st rand-specific. This study presents a simple method for detection and m easurement of PCR bias for any set of primers, and investigates parame ters for overcoming PCR bias.