nested MSPCR and PCR-Bias - (Aug/04/2010 )
I am doing nested PCRs with a first round using BSP and the second round using the methylationspecific primers. This wirks well, however I do not get an visible amplification if I do the MSP without a round of BSP.
Is that normal?
Do I not have to fear PCR-Bias, because the problem in BSP (eg methylation-independent PCR) preferably amplifies the unmethylated allel, if there is one. Could I not get a false negative result for the methylated allel?
What is your experience?
Thanks for your thoughts, Rudi.
In theory, your BSP primers should not induce bias, it would sound like your starting material is quite minimal and that you are using a bisulphite conversion method that is destructive to the sample.
have you tried doing two rounds of MSP?