Help: about the MSP result ! - how to explain the My MSP result? (Jun/28/2005 )
I used a two-stage nested PCR to determine the promoter methylation of a tumor suppressor gene. There is no CpG "C" in the external primers. and the the first step PCR product was diluted 1:50. The internal primer is MSP primer with 3~4 CpG "C" .After the second step PCR ,I obtain the correct MSP product. BUT I have a trouble : the Mehtylated primer and the Unmethylated primer all have PCR product. (I use cancer tissue.amplicons of the Unmethylated primer are expected to be not exist).How to explain the result?
I would say your result is expected. The reasons are:
1) Cancer tissues or cells are not necessarily fully methylated.
2) If PCR conditions are not optimized, either MSP or USP primers may give non-specific amplification.
So your result can be explained as the result of partial methylation or non-specific amplification.
Verify your result by sequencing the product amplified by the external priemrs.