Methylation specific PCR - Methylation specific PCR - problem with controls.... (Sep/18/2009 )
I have recently started doing MSP and I am having problems with my positive and negative controls that I am hoping someone may be able to help me with.... So, I am using universally methylated and universally unmethylated DNA as my positive and negative controls. I bisulfite treat both sets of DNA separately and go ahead with the MSP. When using methylated primers with the bisulfite treated methylated DNA as template I get a really strong product of the correct size. However, the problem is that I also get a product when I use the unmethylated primers with the bisulfite treated methylated DNA as template.... Sometimes this product is smaller than the specific product but in other cases it is of the correct size.... I also have this problem when using the opposite combination of primers and template...The strange thing is that my methylated and unmethylated primers can distinguish between methylated and unmethylated products in cancer cell lines and fresh tissue...
I initially optimised the primers on bisulfite treated DNA from different cell lines and these all worked great. Since I have been getting these problems with the controls I have tried increasing the annealing temp to get rid of the non specific product... I have also made new primer stocks and new bisulfite treated controls in case of cross contamination.... But I am still getting the same problem...
As I am new to this area I can't think of what else I can do to solve the problems I am having (primer design should be ok as designed them using methprimer after I identified the CpG islands in the promoter sequence).
I hope someone can help me, thanks!!
try reducing the number of PCR cycles in your PCR.
methylnick on Sep 18 2009, 11:54 PM said:
Thanks for your help. I have reduced the number of PCR cycles and also increasing the annealing temp but I am still getting a band with the universally methylated DNA and the unmethylated primers.... I have been trying to optimise all PCRs so that they can run at the same annealing temp for both methylated specific and unmethylated specific (even though based on the primers the annealing temp may vary for the different sets of primers)
Does anyone else doing MSP run the methylated reaction at a different annealing temp to the unmethylated reaction????