Pls check my bisulphite sequencing primers - (Jun/30/2005 )
I have gone down as low as 48C in my actual PCR conditions with calculated Tm of primers at 55C to get a product.
that is awesome news, I hope you get some nice results out of it!!!
I'm running into some difficulty selecting primers to amplify the 3 CpG islands in my gene of interest. I'm a little concerned that of the 3 islands that CpG island searcher found, only 1 actually lies 5' of the first exon, which I'm assuming to be the promotor. The 2nd and 3rd actually extend into the first & second exon as per the Ensembl db gene info. Is it even worthwhile to look at the 2nd and 3rd islands?
I'm attaching the mock bisulfite treated DNA if you have some time to take a look at it. My main issue is that I can't seem to find a location to bind my primers to yet still maintain a high Tm and primer size yet also maintain a low product size (between 300-500 bp). Since the first island is about 500 bp I was considering splitting the island into two products and amplifying them separately but I'm not sure if I would really want to do that. Any ideas?
Just to reiterate, I am doing melting curve analysis of 5mC content so I need to amplify the entire CpG island.
Red = CpG
Yellow = CpG island
Pink = Non-CpG converted to Ts
Bold = Exons
it maybe worth your while to have a look at the other CpG islands found downstream of the predicted promoter island just to see how they behave, I would be interested to know what the methylation status of these are because they are non-traditional CpG islands (supposedly not associated with CpG islands and should be hypomethylated. They very well maybe hypermethylated as they may not have a function with respect to the gene).
As for your primers, might I suggest you perform a nested PCR experiment using two sets of PCR primers and then perform a melting curve analysis on the product obtained from the secondary PCR reaction?
The outer primer pair can amplify anything up to 1-1.5kbp with the internal set amplifying your region of interest.
I have modified your first primer set in a manner that will amplify fully converted template and calculated the Tm to be about 62C with perlprimer.
Please have a look at the attached document. and please have a look at primer design in other posts I have made.
There will be inherant bias in fully converted DNA molcules going this route which may affect your melting curve analysis, however it should not be a problem if you perform the same PCR for your standards.