A. General consideration for primer selection for bisulfite-conversion based PCR methods

Because the two strands of DNA are no longer complementary after bisulfite modification, strand-specific primers are used for PCR amplification. Usually the sense strand is chosen for primer design. Primers should be designed according to the guidelines described by Clark and colleagues, and under the assumption that all cytosines had been converted to uracil.

Important parameters to be considered for selecting PCR primers are the ability of a primer to form a stable duplex with a specific site on the target DNA and no duplex formation with the other primer or no hybridization at any other target sites.

Despite variations among bisulfite-conversion based methylation mapping methods, they all share the same procedure of modifying DNA with sodium bisulfite as the first step followed by PCR amplification using primers specific for the modified DNA. The following criteria are common to both methylation specific PCR (MSP) and bisulfite genomic sequencing PCR (BSP).

1. In normal cells, most CpG sites are methylated except those that are within CpG island regions which are defined as DNA stretches at least 200 bp long with a GC content > 50% and an Obs/Exp ratio of CpG dinucleotides > 0.6. More than half of mammalian genes have CpG islands within their 5' regulatory region. In cancer cells or some other situations such as imprinted genes, CpG islands become methylated leading to transcriptional silencing. Thus, for methylation mapping, it is important to focus on CpG island regions.
2. To discriminate between the bisulfite modified DNA and the unmodified DNA, primers should have an adequate number of "C"s in their sequence.

B. Bisulfite sequencing PCR or restriction PCR.

Reference

Li, LC and Dahiya, R. Bioinformatics, 2002; 18(11):1427.