Figure 2

Bisulfite conversion and long-range amplification. (A) Six commercially available bisulfite conversion kits were tested and treated DNAs were examined using an Agilent 2100 Bioanalyzer to assess size distributions. (B) Two bisulfite-converted DNAs from each of the six kits were subjected to PCR with two amplicons (655 and 1109 bp) and three different extension temperatures (65°C, 68°C and 72°C) to assess capacity for long amplicon amplification. Bisulfite-converted DNA from two kits (Epigentek Methylamp and Qiagen EpiTect) with a PCR extension temperature of 65°C had the most robust amplification of the longer 1109 bp amplicon. (C) Eight amplicons ranging in size from 655–4027 bp (overlapping the MEST CpG island) were designed to determine the upper amplicon size limit of bisulfite PCR. (D) Agarose gel image of bisulfite PCR with the eight primer sets using DNA converted by the Epigentek Methylamp and Qiagen EpiTect kits revealing stable amplification of the 1631 bp amplicon with the Epigentek Methylamp converted DNA and the reported PCR conditions.