PCR with 3' mismatch

Wed Aug 31 16:30:24 EST 1994

m-brazil at nimr.mrc.ac.uk (Melanie) wrote:

> Regarding primers with a mismatch at the 3' end. Has anybody used this
> method to distinguish between the presence (or absence) of point mutations?
> Any tips greatly appreciated.

We messed around with this for a long time and it's a bear.  The primers tend
to prime in spite of the mismatches.   We could get preferential priming by
cranking the annealing temp. up to its maximum workable value; but we never
completely suppressed the false positive signal.  I don't recommend this
approach.   My understanding is that LCR has much higher specificity, although
we haven't tried it.

Steve Hardies, Dept. of Biochemistry, Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu

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