Hepatitis C Virus Heteroduplex Tracking Assay: Application to Genotype Determination, Quasispecies Analysis, and Molecular Evolution Studies

The heteroduplex tracking assay (HTA) is a tool that can be used for determining genotype, quasispecies analysis, molecular evolution, and epidemiological studies (1 –7 ). By hybridizing a labeled, single-stranded DNA probe to colinear, reverse transcriptase (RT) PCR products from a sample of interest, the probe will either form a homoduplex with identical molecules or a heteroduplex with nonidentical sequences. The hybridization products are separated on MDE or polyacrylamide gels and visualized. Delwart et al., the developers of the HTA technique (1 ,2 ,7 ), have previously shown that the migration of heteroduplexes relative to the homoduplex on gels are approximately proportional to the percent nucleotide divergence between two species, and therefore, the genetic distance between two species can be determined. Genetic rearrangements, deletions, and/or insertions can alter the migration of heteroduplexes in a manner that disturbs the direct relationship between relative migration and genetic distance. Typically, heteroduplexes of 0.176–1.8 kb containing >1.4–3% to ~30% nucleotide substitutions, which lack genetic alterations, can be identified as unique species on MDE gels (1 ,4 ,6 ). The number and distribution of unique bands indicates the genetic complexity of viral species in each sample.