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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.