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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