Methylation of DNA at carbon 5 of cytosine occurs in almost all living organisms and is a basic element of epigenetic mechanisms that control various genomic functions.
In mammals abnormal methylation profiles of cytosines within CpG islands are present in many disease states, such as cancers. An increasing interest and volume of research in this field has created the need for a quick and reliable method of testing the DNA methylation status. There are many micro or macro array-type techniques that allow genome-wide screening of a methylation profile, yet any result obtained that way has to be validated by detailed analysis of the defined gene. For that a ‘classical’ bisulfite DNA modification method remains unsurpassed.
In this method, the bisulfite reagent converts unmethylated cytosines into uracils and methylated cytosines stay unchanged. This way bisulfite treatment produces sequence differences that can be detected either by methylation specific PCR (MSP), or by sequencing. Since the conversion process lasts several hours, this method is considered time-consuming and technically challenging, but with the MethylDetector™ Bisulfite Modification Kit from Active Motif, it’s easy and results are reliable. It takes merely 1 hour of bench work in total if the conversion reaction is set up for overnight incubation: 10 minutes to prepare reagents and DNA, 9 hours incubation at 50ºC, then not more than 30 minutes the next morning for desulfonation and purification of converted DNA. The kit provides an almost complete set of reagents and the purification columns needed for modification of 50 samples. Since the recommended amount of DNA is 0.2 to 2.0 ug in a volume up to 20 ul per sample, the kit allows for conversion of 100 ug DNA in total. The Conversion Reagent and Buffer, Hydroquinone and Desulfonation Reagent are each aliquoted in 5 tubes; each tube has enough reagent for 10 reactions, which allows economical use. A positive control is also included: PCR primers specific for converted human p16 DNA. This control is very useful as it allows any obtained data to be attributed to a real DNA sequence rather than to false positives or false negatives resulting from the method itself. The vendor also provides information and a link where one can obtain the information on the primer design, which I found very useful.
The instruction manual is clear, consistent and easy-to-follow; it contains almost all information needed. What has been forgotten is information on the DNA polymerase in the section “Additional Materials Required”. I had to call the company to learn what type of polymerase the kit was optimized for.
In my research, I’m looking for methylation of a broad group of genes involved in the early stages of lung diseases. So far I have used the MethylDetector™ Bisulfite Modification Kit to bis-convert about 40 samples of DNA extracted from human tissues as well as from cultured cells. Based on my experience with the kit, I would recommend careful estimation of the starting DNA concentration as it is not possible to predict an extinction coefficient of modified DNA; hence, it’s not possible to assess its concentration after final, on-column desulfonation and purification. Also, going beyond the optimal 2 ug of DNA per reaction may result in insufficient modification which negatively affects further analyses. When I want to modify more that 2 ug of DNA, I simply aliquot my sample into 2 or more and use the appropriate number of tubes and columns.
Overall, I highly recommend this kit to beginners for its ease and clarity, as well as to more advanced researchers for the sake of time saving.