Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications

TLDR

Hot Start PCR delays reagent mixing until 60–80 °C, and the AmpliWax vapor barrier uses a solid wax layer to keep reagents separated until heating melts the wax. Hot Start PCR with an AmpliWax vapor barrier permits routine amplification of a single target molecule, with detection by ethidium‑stained gel electrophoresis and confirmation by nonisotopic probing. Wax‑mediated Hot Start PCR markedly improves specificity, yield, and precision for low‑copy‑number HIV targets, eliminating mis‑primed sequences and primer oligomers even in the presence of human DNA, with benefits evident for copy numbers below ~10³.

Abstract

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 degrees 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 microgram of human placental DNA (1.6 x 10(5) 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; nonisotopically visualized probing suffices for confirmation. The improved amplification performance is evident for target copy numbers below approximately 10(3).