COMBATING PCR BIAS IN BISULFITE-BASED CYTOSINE METHYLATION ANALYSIS -BETAINE-MODIFIED CYTOSINE DEAMINATION PCR

Sodium bisulfite-induced cytosine deamination/PCR (CD-PCR) is currentl y the most sensitive and robust method to determine the methylation st atus of all cytosines in a specific DNA sequence. The CDPCR products a re directly sequenced with Thermosequenase and capillary electrophores is; peak areas are then used to determine the mole fraction of methyla ted cytosines at each site in a single analysis. Here we show that, if the original DNA sample is a mixture of methylated and unmethylated D NA, conventional CDPCR discriminates against the sequence originating from the methylated DNA; CDPCR product does not accurately represent t he methylation status of the original DNA sample. While CDPCR bias can lead to serious errors when determining methylation levels, the addit ion of betaine (N,N,N-trimethylglycine) to the PCR reaction buffer red uces this bias to less than 10%.