MSP Primer Sets (Herman Papers) - (May/23/2006 )

I've been trying to utilize some of the published p16 primer set in JG Hermans methylation paper (Carcinogenesis, vol 24 no. 2 pp 193-198, 2003) but have been running into a very odd problem. I've done my bisulfite treatment and tried to run the nested PCR, but although I get the desired product I also get a lot of "junk" bands and smearing. I've tried to adjust the annealing temperature up and down by running a gradient, but at all annealing temperatures that I tested from 55-65 I got the same "junk" my no template control has nothing. Has anyone encountered problems like this with primers in published papers?

I noticed that almost none of the antisense primers which Herman lists ends on the 3' end with a conversion event or is this something that is more important on the sense primer? Could I be seeing some sort of unspecific binding due to that? Also I saw in his paper that Herman does his bisulfite treatment with 1 ug of DNA. Runs his first round PCR with 1 ul for 30 cycles, and dilutes his first round product 1:1000 to run his nested MSP. Is this typical?

I do my bisulfite treatment with 1 ug of DNA, then run my first round of PCR, then take 1 ul of the first product and run the nested PCR. Could it be that I'm somehow using TOO MUCH template?

cancergeek,

it is possible you could be using too much template, if you are using Herman's protocol/primers it would be wise to follow exactly what Herman did.

I have tried some published primers in the past, and I have encountered typos and sequence errors that could be easily overlooked when publishing a paper, but can be devestating to someone who wants to replicate the data. A simple check to see that the primers match the p16 promoter region will tell you if the primers are correct or not.

Good luck!

Nick