QUANTITATIVE MULTIPLE COMPETITIVE PCR OF HIV-1 DNA IN A SINGLE REACTION TUBE

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 b p) or shorter (122 and 105 bp) than the 142 bp of the wild-type PCR pr oduct. Varying amounts of each of these competitors are introduced tog ether with the sample into a single reaction tube. Since competitors a nd wild-type fragments share the same primer recognition sequence (SK6 8/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 vide o densitometer and calculating the equivalence point in a linear regre ssion plot.