Degenerate Primers - (Mar/14/2007 )
There is definately a bias introduced by some degenerate primers, it is possible that this is related to the initial mixture produced and Davo's comment is a good way to fix that, however I think that there can also be differences in amplification efficiency for the two primer pairs. The experiments that showed this (if you need I can look up the paper, just ask but I won't take the time to do that now) used mixtures of completely unmethylated DNA and compeletely methylated DNA and showed a difference in amplification efficiencies that was improved with higher annealing temperature or touchdown PCR. However it is possible that this bias could only occur when the whole template is differentially methylated though so it may not be relevant to "real life" that depends on your theory of methylation, if one site is differentially methylated are the other sites also differentially methylated? (something like linkage disequilibrium) This may mean that regardless of your approach to primer design the two templates would be differently amplified... I personally don't think there is evidence either way so I would suggest eliminating the variability as much as possible by trying to find primers with no CpG site...
HTH-GL
HTH-GL
Thank you, I think I will try mixing the two types of primer for the bisulfite PCR. And I was also wondering about the problem you mentioned, "if one site is differentially methylated are the other sites also differentially methylated?". So does this mean the problem cannot be solved unless we know the methylation stuatus of the CpG site within the primer or even if we know? For example, if one has sequenced -500 to -300 upstream of TSS using nondegenerate primers and found that the % methylation of a particular CpG site at -330 is ~70%, then he wants to check the methylation status of -300 to -100 and he designs a forward primer which includes the CpG site at -330, do you guys think he should mix the methylated primer (primer with C at the CpG site) and the unmethylated primer (primer with T at the CpG site) in a ratio of 7 to 3 or 5 to 5?
The sequence directly upstream and downstream of the TSS of my target gene is very dense in CpG sites, I designed different primers to amplify the region, both degenerate and nondegenerate, but to no avail. I first tried the nondegenerate primers, but there was no PCR product. I thought maybe the primers are too short, only 16-18nt. Then I lengthened the primers (up to 27mer) and as a result, degenerate sites are unavoidable, the PCR didn't work either (temperature gradient/touchdown with/without DMSO, Betaine or Mg2+ titration had been attempted). After that, I designed another three sets of primers (also degenerate) further away from this GC rich region, the PCR worked in the first attempt but led to another problem which I have described previously in this thread.
I wonder why bisulfite PCR could be so difficult...
Sorry to have confused you, I actually mean that the amplification efficiency differs between the fully methylated and fully unmethylated template simply due to differences in GC content of the amplimer, and if your test amplicons are not essentially equal in GC content you may see these amplification differences (due to the GC content of the amplimer, not due to the primers) So I think you should stick with a 50-50 mix for the primers... To help solve your problem I would suggest using a touchdown PCR, it will aid in specificity and was also found to correct the above mentioned amplification differences for methylated vs unmethylated DNA...
Hope this helps you, and Good Luck!