Nucleic Acids Research, 1992, Vol. 20, No. 7 1717-1723
© 1992
MOLECULAR BIOLOGY |
Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications
Cetus Corporation 1400 Fifty-third Street, Emeryville, CA 94608, USA
*To whom correspondence should be addressed at Applied Biosystems Inc, 850 Lincoln Centre Drive, Foster City, CA 94404, USA
Received November 14, 1991. Revised February 25, 1992. Accepted February 25, 1992.
A Hot Start Polymerase Chain Reaction (PCR) entails the withholding of at least one reagent from the reaction mixture until the reaction tube temperature has reached 60–80°C. Hot Start amplification with an AmpliWax vapor barrier uses a layer of solid wax to separate the retained reagent(s) and the test sample from the bulk of the reagents until the first heating step of automated thermal cycling melts the wax and convectively mixes the two aqueous layers. Wax-mediated Hot Start PCR greatly increases the specificity, yield, and precision of amplifying low copy numbers of three HIV targets. In the presence of 1 µg of human placental DNA (1.6 x 105 diploid genomes) the specificity improvement entails considerable to complete reduction in the amplification of mis-primed sequences and putative primer oligomers. When mis-priming is negligible, the procedural improvement still suppresses putative primer oligomerization. Hot Start PCR with an AmpliWax vapor barrier permits routine amplification of a single target molecule with detection by ethidium stained gel electrophoresis; noniso-topically visualized probing suffices for confirmation. The improved amplification performance is evident for target copy numbers below approximately 103.
Present addresses: Roche Molecular Systems, 1145 Atlantic Ave, Alameda, CA 94501, USA
Present addresses: Berlex Biosciences, 213 E. Grand Ave, South San Fransisco, CA 94080
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