A method for nested PCR with single closed reaction tubes.
Toward the goal of reducing diagnostic false positives while retaining high sensitivity, a closed-tube nested PCR procedure has been developed for detecting low-copy-number human immunodeficiency virus (HIV) gag target DNA sequences. Master mix for amplification 2, in a hanging gel matrix at the reaction tube top, remains sequestered from the reaction space of the tube during amplification 1. A severalfold excess of inner over outer primers is built into the procedure to assure the high sensitivity of nested PCR. The master mix for amplification 2 is then introduced into the reaction space by centrifugation, and the second amplification is performed as usual. The closed-tube nested procedure shows sensitivity approaching that of the open-tube control procedure, which detects a single copy of HIV gag target DNA at near-theoretical frequency, typically with microgram yields of specific amplification product.
Yourno
J
J
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D015750:Genes, gag; D006678:HIV / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*
D004279:DNA, Viral