Differential PCR based on 3' mismatch
jkropp at itsa.ucsf.edu
Fri Jun 13 19:25:39 EST 1997
In article <9706128660.AA866090364 at smtpgwy.agric.nsw.gov.au>,
deborah.hailstones at SMTPGWY.AGRIC.NSW.GOV.AU ("Deborah Hailstones") wrote:
> Dear all,
> We have been trying to use PCR to differentiate between two closely
> related viroids, which differ from eachother by only a few
> nucleotides. We have designed PCR primers to these regions, placing
> the differences at the 3' ends of the primers, to try to distinguish
> between the viroids. Sounds great on paper but the primers don't
> actually seem to distinguish between them at annealing temp Tm - 5
> oC.... it seems very odd that there is no specificity. Surely this
> runs against the accepted wisdom of how PCR works? Does any one out
> there have experience wiht this sort of assay? we are at a loss to
> explain it...
Lillian Kwok, back when there was a Cetus, did some studies on 3' mismatches.
I don't have the refs now, but you should be able to find them. I remember
that she found a G-T mismatch to be the most likely not to impede primer
extension. However, in the one instance when I tried it, that very
mismatch did discriminate between two alleles. That is, I got extension
only with the perfect match.
Still, you can probably increase your chances of discriminating by
following her guidelines. Also, some people have suggested making the
mismatch at the penultimate base instead.
Cosolvents such as DMSO, TMAC, or betaine may help.
You can do some online primer evaluation and Tm at the following:
Primers are cheap these days. Try several if you have the strength.
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