Background: The Celera Diagnostics (formerly Applied Biosystems) ViroSeq HIV-1 Genotyping System is a commercial system for analysis of drug-resistance mutations in HIV-1. This system uses population sequencing to analyze protease and RT sequences in DNA amplified from plasma HIV-1. A dUTP/uracil N-glycosylase (UNG) contamination control system is incorporated in the system to prevent PCR carry-over. In this system, dUTP is incorporated into PCR products in place of dTTP. This complicates cloning of the PCR products, since most strains of E. coli contain the UNG enzyme, which destroys dUTP-containing DNA.

Methods: We developed a novel approach for cloning gag-pol HIV-1 DNA produced in the ViroSeq system. To prevent degradation of dUTP-containing DNA amplified in the ViroSeq system, we cloned the PCR products into a commercial cloning vector, and transformed the ligated plasmids into UNG-negative E. coli. For these studies, 3 different strains of UNG-negative E. coli were rendered competent for electroporation, and the transformation efficiencies of the 3 strains were compared. One (1) strain, available through the E. coli Genetic Stock Center at Yale University, was superior to the other strains tested. Plasmids were isolated from the transformed bacteria using a commercial DNA prep kit. Protease and RT sequences were obtained using the ViroSeq sequencing module and analyzed with ViroSeq software. This system was used to clone and analyze HIV-1 from clinical trial samples using PCR products remaining from a previous genotyping study.

Results: PCR products produced during amplification in the ViroSeq system were successfully cloned using as little as 1 microliter of the amplified DNA that remained after routine genotyping. Analysis of 10 clones from each sample provided information about the genetic linkage of resistance mutations, and revealed the presence of mutations not detected by population sequencing.

Conclusions: The methods described above were used to clone dUTP-containing gag-pol DNA amplified in the ViroSeq system during routine genotyping. This approach does not require additional plasma for cloning. Cloned plasmids are derived directly from the PCR amplicons used for population sequencing. Analysis of cloned HIV-1 variants using this approach can provide information about the genetic linkage of drug resistance mutations and may identify minority variants not detected by population sequencing.