spotlight question - (Jan/17/2007 )
I have the problem I can not menage with, I am working on methylation and so far everything was perfect I found the methylation ( MSP confirmed by BSP) in the cell line without the protein expression when I treated the cell line with 5 aza the expression on m RNA level was restored but when I am taking DNA on the BSP (10 clone sequenced I do not see any drop in methylation) whereas when I am doing the MSP (my cell line which are methylated have two bands on MSP and it was confrimed by BSP that template for both primers exists) I see that methylated band is weaker whereas unmethylaated stays the same. I also had a problem to create the BSP product from DNA t\from cell line treate dwith 5 aza. after PCR I have much less product that in control (not tretaed with aza dna)
so my question is ;Is it possible tjat duriong creating BSP product in some way I select only for the better quality DNA so DNA from the cells which did not incorporate 5 aza during treatment and because of this I see the restoration of expression but not change on DNA level???
the problem which you are facing can be explained by the specific mode of action of 5-AZA. It is incorporated into newly synthetized DNA and covalently binds DNMTs. So, if you do not wait for at least two cell cycles, you will find a lot of hemimethylated DNA, which you cannot distinguish from fully methylated DNA. How long did you incubate your cells?
I'll paste a brilliant review by Judith Christman on that topic, this should explain it.