Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA.
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.
Warnecke
P M
PM
Kanematsu Laboratories, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia, School of Biological Sciences, A12 University of Sydney, NSW 2006, Australia and CSIRO Division of Molecular Science, Sydney.
Stirzaker
C
C
Melki
J R
JR
Millar
D S
DS
Paul
C L
CL
Clark
S J
SJ
D044503:5-Methylcytosine; D000818:Animals; D001482:Base Composition; D002460:Cell Line; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D017931:DNA Primers; D016161:Genes, Retinoblastoma / Q000235:genetics; D006801:Humans; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013447:Sulfites
D017931:DNA Primers; D013447:Sulfites; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA