some questions in DNA methylation - (May/16/2006 )
Using MSP, I found one gene is totally methylated and the other gene is totally unmethylated. But I found the third gene is hemimethylated because I can get the PCR products using both U and M pimers. The DNA treated with bisulfite is the same.
I am not sure the hemimethylated gene and whether it happens for a gene to be partly methylated in rat cell line. Someone said that hemimethylated gene is seldom detected in adult human samples. I am wondering whether rat cell line is the same situation.
Another question is whether it is always necessary to include the negative control (unmethylated genome) in MSP or BSP. Because negative control DNA is difficult to get for rat or mouse genome, ( for human, it can be got from chemicon corp.), it is good to only use positive control or to use human meth- and unmethylated genome as controls?
Thanks for your reply!
Hmmm, I'm not sure what tissue you're using from rats or rather where you're genomic DNA is coming from in the rats. Maybe lymphocytic DNA? But any normally expressed gene should be unmethylated. Especially a tumor suppressor gene unrelated to what you're studying. Those promotor regions can serve as a negative control. I don't know if a negative control is essential for MSP or BSP. For a negative control you can just pick a gene and design a pair of primers.
I see. Thank you.
I use normal Taq polymerase and get the PCR bands. There are always only two bands in my DNA gel which are a little bit close. According to MW, I can decide which band is I want. But it might be better if I use hotstart Taq polymerase. I never use such kind of Taq. Recently, I used hotstart polymerase (95 degree, 15 min). but the result was awful and it seemed no band detected. I am not sure wether the hotstart Taq is used correctly. Could you tell me?
Because I just set PCR cycler as 95 degee, 15 min but not add hotstart Taq after reaching 95 degree.