Taq slippage in PCR

John H McDonald mcdonald at strauss.udel.edu
Mon Oct 3 18:18:51 EST 1994


I'm trying to PCR and then directly sequence some DNA with a couple of
runs of 7 to 10 T's.  I'm having problems, because "slippage" causes
the number of T's to be variable within a single PCR product.  I suspect
this problem occurs during the PCR, since it is worse when I try to
sequence PCR products that are reamplifications rather than amplifications
straight from genomic DNA.  Slippage during sequencing may also contribute
to the problem, though. 
	I know the problem of slippage has been discussed before here with
regards to dinucleotide repeats, but what about single-base runs?  Aside
from keeping the number of rounds of PCR to a minimum, has anyone found a
cure for this problem?  Has anyone tried things like increasing the
annealing temperature, reducing the annealing time, decreasing the
magnesium, changing the pH or other buffer constituents, and convinced
themselves that it DIDN'T help?  (This would be useful to know, so
everyone who runs into this problem won't try the same failed experiments.)

John H. McDonald
Department of Biology
University of Delaware



More information about the Methods mailing list