title: Low-concentration initiator primers improve the amplification of gene targets with high sequence variability.
abstract: The amplification and detection of diverse strains of an infectious virus or bacteria, or variants within a gene family is important for both clinical and basic research but can be difficult using conventional PCR. This report describes and illustrates a novel closed-tube method for amplifying and characterizing heterogeneous target sequences using members of the CTX-M beta-lactamase gene family. Different subgroups of CTX-M genes exhibit low sequence identity, but accurate and efficient detection of these variants is critical because they all confer resistance to penicillin, cefotaxime, and other antibiotics of the beta-lactam class. The method combines a single pair of "thermodynamic consensus primers" (tcPrimers) with one or more "initiator primers" (iPrimers), added at low concentration (5-10 nM). Each iPrimer improves the initial amplification of one or more variants because it has fewer mismatches to its intended target than the more abundant tcPrimers. As a result of initial amplification, each heterogeneous sequence is shifted stepwise toward a better match with the tcPrimers. As soon as the tcPrimer hybridization takes place, amplification proceeds with high efficiency. The tcPrimer pairs can be designed for symmetric PCR or for Linear-After-The-Exponential (LATE)-PCR. LATE-PCR offers the advantage of generating single-stranded DNA that can be characterized for different gene variants in the same closed tube, using low-temperature mismatch-tolerant fluorescent probes.
authors.affiliation: Department of Biology MS-008, Brandeis University, Waltham, MA, 02454-9110, USA, pierce@brandeis.edu.
authors.forename: Kenneth E
authors.initials: KE
authors.lastname: Pierce
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Wangh
mesh_terms: D017931:DNA Primers / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers