BSP Primer placement over KNOWN unmethylated CpG's - Can this be done? (May/02/2006 )
Quick question about primer design in BSP...
So I've sequenced the 1st 500bp in my contiguous 1300 bp promoter region. I'm designing the 2nd pair of primers and of course, I want them to overlap with previously sequenced area.
My dilemma is: Can I design my forward primer (which will be in the overlap area) over CpG's. Normally I know that you're not supposed to. But in this case I've already sequenced that area where the forward primer is going to be placed. Therefore I already know the methylation status of those particular CpG's. Those CpG's are not methylated and were converted from C's to T's in the bisulfite reaction according to the ealier direct sequencing data. So I think there is no harm in having some CpG's in my forward primer. I'm not sure though. The reason I would like to do this is because there are so many CpG's (in that overlap area) that there is hardly any stretch of 20-30 bp's where I could place the forward primer.
If anyone has any ideas or thoughts, appreciate it.
I have done so in the past and I try and pick the primer such that the CpG's are mainly at the 5' end and avoid them being in the 3' end of the primer as the 3' end is the most discriminating part of the primer in terms of DNA sequence. Having said that, I usually pick wobbles at the C's of CpG's as for me, the methylation status is unknown.
In your case purplefetus, I suppose it would be okay to do so, but there will be a risk of induced PCR bias by doing this and you should be aware of it, granted primers picked for MSP and BSP will induce bias to fabvour converted DNA, by selecting primers with CpG's that are not methylated, you will be selectively amplifying the unmethylated population of your gDNA which may not be ideal. I have successfully amplified and cloned a 1.2kb fragment and there are papers on MDR1 which have done so too, maybe you could do the same, to avoid this situation.
Wow, thanks Nick. I have to avoid introducing this type of bias. I guess I will not put any CpG's in my primers and risk using a shorter primer (~20bp's instead of the normal 25-30bp's) I was not aware of the selective amplification I would be inducing. Appreciate the info. And unfortunately our sequencing center only can sequence up to 700bp amplicons. So I will have to design 3 sets of primers or maybe 4...we'll see how it goes.
700bp is standard, on a good day I can get up to 1kb of good sequence but I don't get many good days and I am really pushing the assay to the limit then