How stable is methylated DNA?
Posted 02 June 2009 - 06:49 AM
My question may be stupid, but since I'm new at this I'll ask it anyway: how stable is methylation of DNA? I have made DNA preps about 2 months ago, and since then I have been using the EZ methylation kit to try and figure out if there is any methylation in the promoter of my gene. I'm surprised to see all Cs change to Ts in my sequencing results. I've increased the amount of DNA that I treat in case it wasn't sufficient and everything got changed because of that. I'm now at 500ng per reaction, which is the high end of the optimal range recommended.
Also, how frequent is DNA methylation? Is it normal that promoters wouldn't have any methylation at all? I work on Phytophthora sojae, a pathogen of soybean, and unfortunately I can't find any gene that is known to have methylation, to use as positive control. So far I've worked up over 1000bp upstream of start ATG and see no methylation at all.
Thanks a lot
Posted 02 June 2009 - 02:50 PM
HAven't tested the longevity with the Zymo kit, though with the homemade reagents and conversion, DNA can last up to year before I have used it all up.
As for the results you are seeing, I am not familiar with the pathogen, however there are some organisms that don't have much or any DNA methylation at all and could your organism of interest fall into that category?
One area to potentially look at for DNA methylation would be centromeric, satellite or repetitive sequences that are found in your organism.
All comments and communication are my own personal ones, and are not tied to any of my affiliations.
Posted 03 June 2009 - 09:27 PM
500 ng DNA is a good amount for conversion. All C's are converted to Ts, suggesting there is no methylation, although there is slim chance of overconversion. According to the original paper describing the bisulfite conversion reaction, overreaction can also convert methylated cytosine to urcils. You can use methylated DNA as controls.
Posted 04 June 2009 - 01:58 AM
I'm working on epigenetics for approximately 2 years. I've been using EZ DNA kit without any problem. I use 1μg of gDNA for conversion. As far as I know, repetitive freeze/thaw is not the best for bisulfite DNA. That's why I tend to aliquot my DNA in small quantities (5-10μl). I cannot confirm whether aging of the sample contribute to more Ts than Cs (or the opposite), but I was able to discriminate between methylated or unmethylated regions for samples at least 6 months old.
Posted 04 June 2009 - 05:40 AM
I'll try also the first half of the gene (perhaps elongation would be inhibited by methylation) and if not, look in another direction.
Edited by Irina, 04 June 2009 - 05:40 AM.