Research of epigenetic mechanisms has great importance in the area of genetics and molecular biology. One of the most common epigenetic mechanisms is methylation. Naturally occurring methylation has an important role in normal genomic organization, such as imprinting and X-inactivation. Besides this, it is one of the most common gene expression regulation mechanisms in health and disease. For example, aberrant methylation of some genes in many cancer types has been reported. So, one should detect methylation status of a region in order to delineate the mechanism of a disease or many regularly-occurring phenomena. There are some techniques for this purpose, such as methylation-sensitive, arbitrarily-primed PCR. One of the most common techniques is the bisulfite method; there are several kits which use the bisulfate method for detection of methylated DNA. We use Zymo Research’s EZ DNA Methylation Kit™. The kit has two alternative sizes: 50 and 200 reactions. The kit includes CT conversion reagent, M-dilution buffer, M-binding buffer, M-wash buffer, M-desulphonation buffer, M-elution buffer, columns and collection tubes.
The system is based upon converting all unmethylated cytosine residues to uracil using chemical treatment. The technique consists of treating DNA with bisulfate, which converts unmethylated cytosines to uracil, while methylated cytosines remain unchanged. In this procedure, modified and unmodified DNA is amplified by PCR. Detection can be done by restriction endonuclease digestion or direct sequencing. Then, you can ascertain the methylation status of the sample by comparing the sequence of the bisulfite-treated DNA to that of the untreated DNA. The EZ DNA Methylation-Gold Kit™ integrates the DNA denaturation and bisulfite conversion processes into one step. The kit has been streamlined for high yield recovery of DNA following DNA bisulfite conversion.
The amount of DNA per treatment can range from 500 pg to 2 μg; the optimal amount is 200-500 ng. After preparing the CT-conversion and M-wash buffers according to kit’s manual, you add 130 µl of the CT-conversion reagent to the DNA sample and adjust the total volume to the 150 µl with distilled water. Mix by pipetting. Then, incubate the sample at 98ºC for 10 minutes, then 64 ºC for 2.5 hours, and then 4ºC for up to 20 hours. After that, add 600 μl of M-Binding Buffer into a Zymo-Spin IC™ Column which has been placed into a collection tube (provided). Then load the prepared DNA-CT conversion agent sample into the Zymo-Spin IC™ Column containing the M-Binding Buffer. Close the cap and mix by inverting the column several times. Centrifuge these columns at full speed. The next step is adding M-Desulphonation Buffer to the column and letting it stand at room temperature (20 – 30°C) for 15-20 minutes. After the incubation, spin the columns again at full speed for 30 seconds. The next step is washing. By applying the wash buffer and centrifuging, you wash the sample. Repeat this washing procedure two times. The last step is elution of the DNA from the columns. The manual recommends using 10 µl of elution buffer. By centrifuging, you obtain the DNA in a clean tube. We find it convenient to use 2-4 µl of DNA for the PCR amplification step. An important point one should consider is that high input levels (>500 ng) of DNA may result in incomplete bisulfate conversion for some GC-rich regions.
This kit has in-column desulphonation technology which eliminates cumbersome DNA precipitation steps. The kit has some advantages like minimizing template degradation and loss of DNA during treatment and clean-up. From our experiences with this kit, the recovered DNA was successful for PCR amplification and downstream restriction endonuclease digestion. I think the recovered DNA would be suitable for other downstream assays as well, such as sequencing, microarrays, etc. The procedure is less time-consuming as compared to some other DNA methylation kits. It can be completed in approximately 3 hours.