PCR for sequencing genomic DNA (multiple alleles) - Are there preferences for amplification of certain sequences? (Jan/26/2011 )
I'm using PCR to amplify a fragment for sequencing (after subcloning) and I suspect, but don't know for certain, that there may be two alternative alleles (with maybe just a couple of SNPs).
Someone told me that one allele is likely to be favoured over the other, being amplified more efficiently. But I can't see that happening, because by the time the polymerase comes to a partiuclar SNP, it's already in too deep, and not going to detach, surely? I can't imagine it. Are certain nucleic acids more likely than others to cause the polymerase to come off before completion?
Well, if you have a couple of G or C, they would be more adhesive to the complementary strand, and may not come off as quickly as A or T. So you will have relatively more single stranded AT than GC as a template for PCR. But the polymerase doesn't fall off... And usually it shouldn't make a big difference, if your denaturation step is longer than 10sec.