Single-tube nested competitive PCR with homologous competitor for quantitation of DNA target sequences: Theoretical description of heteroduplex formation, evaluation of sensitivity, precision and linear range of the method

Abstract

Competitive PCR is a frequently used technique for quantitation of DNA and mRNA. However, the application of the most favourable homologous mutated competitors is impeded by the formation of heteroduplex molecules which complicates the data evaluation and may lead to quantitation errors. Moreover, in most cases a single quantitation of an unknown sample requires multiple competitive reactions for identification of the equivalence point. In the present study, a highly efficient and reliable method as well as the underlying theoretical model is described. The mathematical solutions of this model provide the basis for single-tube quantitation using a homologous mutated competitor. For quantitation of Human Papilloma Virus 16-DNA, it is shown that single tube quantitations using simple PAGE separation and video evaluation for signal analysis permit linear detection within more than two orders of magnitude. In addition, repeated single-tube competitive PCRs exhibited good precision (average standard deviation 5%), even if carried out as nested high cycle PCR for quantitation of low abundant sequences (intraassay sensitivity < 2 x 102 copies). This evaluation method can be applied to any DNA separation and detection method which is capable of resolving the heteroduplex fraction from both homoduplex molecules.