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PCR for sequencing genomic DNA (multiple alleles)


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#1 seanspotatobusiness

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Posted 26 January 2011 - 01:20 PM

Hi,

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?

#2 Rsm

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Posted 27 January 2011 - 12:06 AM

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.
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