Nested MSP? - (Jan/10/2007 )
I have a question regarding the use of nested primer sets for MSP. Is it better in the first reaction to use primers that do not discriminate between methylated vs. unmethylated DNA? Also, how many cycles do you recommend for the first reaction and the second reaction. Thanks!
it is better to use degenerate primers in the first round.
however I have seen papers that use only one round and obtain an amplicon.
As for number of cycles, the lower the better, because it is likely your sample will have a mixed population of methylated and unmethylated templates due to normal/tumour tissue mixing or the like, and if you saturate the cycles you will get erroneous results in that amplicons are detected for both the M and U primer sets.
15-20 cycles per round is a good starting point.
Thanks Nick. I have been able to do MSP fine with single round of PCR, but for some reason this region has been giving me fits trying to balance sensitivity vs. specificity. I could probably design new primers for single round MSP, but I figure this will be just as valid.
it's certainly a fine balance between the two, MSP is great for screening samples, you may need to do BSP if you want further infomration.