Quantitative Multiple Competitive PCR of HIV-1 DNA in a Single Reaction Tube

Abstract

A quantitative multiple competitive PCR (QMC-PCR) for determination of DNA copy numbers is described. Four competitive DNA templates for the env region of HIV-1 were constructed with sizes longer (187 and 163 bp) or shorter (122 and 105 bp) than the 142 bp of the wild-type PCR product. Varying amounts of each of these competitors are introduced together with the sample into a single reaction tube. Since competitors and wild-type fragments share the same primer recognition sequence (SK68/SK69), amplification occurs according to the rate of the introduced copy numbers. The PCR products are run on an agarose gel, and the copy number of the sample is determined by analyzing the bands with a video densitometer and calculating the equivalence point in a linear regression plot.