Table 3.

Technologies to analyze specific methylated regions

Method	Key features	Advantages	Disadvantages
Methylation-specific PCR (MSP)	DNA primers are designed to distinguish between methylated or un-methylated DNA. Bisulfite-modified DNA is amplified	Very sensitive; will identify very low levels of methylated DNA in a sample	Very sensitive; easily contaminated; requires further analysis to determine level of methylation present
Combined bisulfite restriction analysis (CoBRA)	Bisulfite-modified DNA is amplified using non-discriminatory primers. PCR product is digested with restriction enzymes that are specific to methylated DNA sequences	Robust detection of methylation; not prone to false positive results	Does not give detailed analysis of region amplified; requires complete bisulfite conversion to prevent PCR bias
Bisulfite sequencing	Bisulfite-modified DNA is amplified using non-discriminatory primers. PCR product is cloned and sequenced	Informative for all CpGs within the region; provides allele-specific methylation information	Laborious
Pyro-sequencing	Bisulfite-modified DNA is amplified using non-discriminatory primers and sequenced using pyro-sequencing technology	Multiple samples can be analyzed in parallel; quantitative	Analysis is restricted by small read sizes