MSP bands - both in methylated and unmethylated DNA (Jun/29/2005 )
I have designed several MSP primers (M primers)using Methprimer program.
They gave nice clear single bands. The trouble is they gave bands both for methylated DNA (SssI treated DNA) and normal tissue DNA. Are there any points I should consider before ordering new primers?
could it be that your normal tissue DNA is also methylated?
do your USP primers come up with bands as well?
apart from your normal tissure being methylated as well, I would say that it could be your bisulfite has not worked efficently.
I used the same DNA for another gene using
a published MSP primer set, and confirmed that
the DNA was unmethylated for that gene.
However, I am not sure if the DNA is not
methylated for the gene I am going to study.
Maybe I'll try using U primers or increasing
the annealing temperature a bit and see if
the band disappears.
the "gold" standard for MS-PCR is that your run both MSP and USP primers, so you will have to check that your USP comes up negative and produce these results in a publication.
I think your gene may well be methylated!