Nucleic Acids Research, Vol 25, Issue 3 654-658, Copyright © 1997 by Oxford University Press
C Christopherson, J Sninsky and S Kwok
We investigated the effects of internal primer-template mismatches on the
efficiency of reverse transcription and PCR amplification. As models, RNA
transcripts representative of different HIV-1 group M subtypes were
evaluated with a previously described gag primer pair system. We observed
that the presence of two to four mismatches in the primer-template duplexes
did not have a significant effect on RT-PCR. However, the presence of five
and six mismatches with the 28 and 30 base primers reduced PCR product
yield by approximately 22- and 100- fold respectively, relative to the
homologous template. The amount of reduction was reproducible from
experiment to experiment and was independent of the initial copy number
input. Under the conditions used, viral RNA measurements of the more
divergent HIV-1 subtypes (A and E) would be underestimated, while isolates
of subtypes B, C, D and F-H are expected to be efficiently amplified and
accurately measured. The reduced amplification efficiency for targets
similar to HIV subtypes A and E can be improved 4- to 10-fold by lowering
the annealing temperature and implementing a reverse transcription step
that gradually increases in temperature. The additional substitution of
either 5-methylcytosine for cytosine throughout or the substitution of
inosine at positions of variable bases resulted in a <4-fold difference
in product yield between the homologous and most divergent templates.
ARTICLES
The effects of internal primer-template mismatches on RT-PCR: HIV-1 model studies
Roche Molecular Systems Inc., 1145 Atlantic Avenue, Alameda, CA 94501, USA. cindy.christopherson@roche.com
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