Genotyping HIV-1 and HCV strains by a combinatorial DNA melting assay (COMA).

Human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) strains can be genetically classified into genetic lineages known as genetic types or subtypes according to phylogenetic analyses of complete or partial nucleotide sequences of their genomes. The genetic classification of HIV-1 and HCV strains has important implications for the development of globally effective vaccines and for the management of patients. A new method, termed combinatorial DNA melting assay (COMA), allows rapid accessing of comparative genetic information between related DNA sequences, making it possible to rapidly and accurately genotype unknown HIV-1 and HCV strains. COMA is mainly based on the differential melting properties of long DNA heteroduplexes. Combinatorial arrays of DNA heteroduplexes are formed when captured PCR-amplified reference DNA with known nucleotide sequences are combined with solution-phase complementary and antigenically labeled DNA with unknown sequences. Genetic divergence between the known and the unknown sequences is inferred as the experimentally derived melting curves of the two strands of the DNA heteroduplexes increasingly diverge. COMA was successfully applied to the genetic classification of HIV-1 and HCV strains into phylogenetic lineages or subtypes. Use of this assay should accelerate current efforts to understand the global molecular epidemiology of HIV-1 and HCV and may extend to the genetic characterization of other genetically diverse infectious pathogens associated with numerous diseases.