Strategy for Controlling Preferential Amplification and Avoiding False Negatives in PCR Typing

Abstract

The use of the PCR method for routine testing has increased dramatically during recent years. Most assays involve coamplification either of an internal control, of several alleles at a given locus or of a variety of bands produced by low-stringency primer annealing. In such multiplex reactions, certain products will often amplify preferentially. Amplimers that are more sensitive can be outcompeted under suboptimal PCR conditions, leading to assignment of false negatives. Optimization of PCR parameters such as temperature steps, relative concentrations of primers and their annealing temperature do not alone ensure against false negatives when caused by stable double-stranded DNA (dsDNA) regions in the amplified sequence. A two-step strategy to solve this problem is presented in this paper: (i) titration of the PCR with NaCl as a model inhibitor to establish the critical range within which false negatives occur; (ii) titration of the PCR with a dsDNA-destabilizing additive under false-negative-inducing conditions until the relative amplification efficiencies of coamplified fragments are adjusted. Betaine is introduced as a novel and efficient cosolute. These measures to achieve reliable PCR typing of a difficult target should be useful for many qualitative and quantitative multiplex PCR applications.