Abstract
A DNA heteroduplex tracking assay (HTA) using single-stranded probes is described. This assay provides a rapid means of resolving genetic variants coamplified by PCR and of measuring the level of particular variants in complex populations. To confidently detect minor quasispecies changes, the importance of maximizing template input into nested PCR (nPCR) and of duplicating nPCR and HTA to ensure correct population sampling is highlighted. The sensitivity of detection of rare variants within a genetically mixed population using single-stranded DNA probes is shown to be 1:500. The effects of nucleotide substitution at different locations on heteroduplex electrophoretic mobility are used to illustrate the limits of HTA for mutation detection. This simple assay may be used to track the evolution of HIV as well as to address issues of contamination and transmission.
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