MSP PCR - (Apr/19/2006 )

Hello PCRMAN

I am new to the forum, hope I get my problems solved here.
I am interested in the methylation status of WIF-1 promoter (a Chromosome 12 gene)
I have tried 2 primer pairs from different publications, and with all the possible optimisation to no avail. The only difference is that these Authors used AmpliTaq Gold DNA polymerase, Or hotstar Tag polymerase, and me ordinary taq polymerase, could this make such a difference, that I obtain o results, especially for the M-msp primers.
In ordinary pcr I would test the primers in e-pcr/virtualpcr (in-silico-PCR), I dont know of any such possibility for MSP-pcr. maybe I should just post the primers here for your advice
m-msp Wif-1 [b]F-GGGCGTTTTATTGGGCGTAT R-AAACCAACAATCAACGAAC 60 ◦C/35cycles
u-msp Wif-1 F-GGGTGTTTTATTGGGTGTAT R-AAACCAACAATCAACAAAAC 60 ◦C/35cycles

I hope this information is enough to fetch some helpful information for me.
Thank you very much for your attention

hi wanyu,

one of the things do with caution is to use primers within a publication, there is lots of room for typo errors and you can never tell if there is one, and if so, it would be even more crucial in an MSP primer set as to whether they work or not.

Have you downloaded the promoter sequence and seen that these primer sequences are there? Remember, when you download the sequence, you must "mock convert" ie: change all non-CpG C's to T's and then your primers will be revealed.

See how you go.

Nick

Thanks pcrman
With your advice I just discovered that the primers were not of the right sequence. Just done the necessary modifications and placed an order for the new oligos, hope it works this time around.
Wanyu