Table 3 Technologies to analyze specific methylated regions
From: Epigenetics of renal cell carcinoma: the path towards new diagnostics and therapeutics
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 |