This review discusses the advantages of MethyLight, a real- time PCR based method for DNA methylation quantitation from bisulfite- modified samples, and compares MethyLight to other related procedures like bisulfite sequencing, MSP, MS-SNuPE, and COBRA. First, as MethyLight is based on real- time PCR, there is no risk of contamination from input DNA or from handling errors. A limitation for this implementation is that MethyLight only detects a particular methylation at a time, but this can be corrected by automation, via simultaneous analysis of many samples in microtiter plates. The detection of CpG hypermethylation is better performed in this manner:
Principles of MethyLight technology (Trinh et al./Methods)
Authors discussed each step for this procedure: (1) determining the site of interest for methylation analysis; (2) methylation-specific primers and fluorogenic probes design; (3) genomic DNA isolation; (4) bisulfite conversion; (5) real- time methylation analysis; and (6) data processing. For primer design, some complications particular to the bisulfite modification should be taken into account:
All unmethylated cytosine residues in the genome are converted [...;] this reduction in genomic complexity reduces the annealing specificity of PCR primers and fluorogenic probes, which not only complicates primer and probe design, but also reduces the efficiency of each PCR cycle. Therefore, PCR amplification of bisulfite converted DNA requires more cycles, and is much less efficient than amplification of [... unmodified] DNA. Essentially, the amplification is being performed on a 3-base genome [...;] furthermore, amplification [...] is inefficient [... because] the template consists of a mixture of different DNA methylation patterns.The amplicon typically should be 50-200 bp long, but if longer amplicons are desired, probes should be designed as close to the primer as possible. This probe is usually linked to a 5' reporter (mostly FAM) and a 3' quencher (e.g., TAMRA). Thus, a polymerase with 5' -> 3' exonuclease activity is required to process the reporter. On the other hand, respect to the sample DNA isolation, it does not need to be extensively purified as the bisulfite modification removes most contaminants -although in some cases these bisulfite chemicals are inhibitors of the PCR amplification. However, it should be kept in mind that very long bisulfite incubations produce more DNA degradation, and that even the sample storage is crucial since bisulfite- modified DNA is unstable. In conclusion, MethyLight has some drawbacks, but is very useful because it is able to process many samples in short time, can be automated, and does not require post- PCR manipulation; therefore is very practical for rapid screenings and other similar applications. The original article that introduced this method will be commented here later.
Trinh, BN. et al. 2001. Methods 25(4):456-462
epigenetics
