Heteroduplex mobility assay for subtyping HIV-1: improved methodology and comparison with phylogenetic analysis of sequence data.
The feasibility of using DNA heteroduplex mobility analysis (HMA) as a rapid and reproducible method for routine subtyping HIV-1 in clinical specimens was examined by comparison with subtype determination by sequencing in both the gag and env genes. The heteroduplexes formed were examined by conventional polyacrylamide gel electrophoresis (PAGE) and also by electrophoresis in the Pharmacia PhastSystem. The significance of the HMA results was determined by the Kruskal-Wallis test, a non-parametric one way analysis of variance. It was possible to obtain an HMA profile rapidly (1-2 days) using fast PCR conditions and the PhastSystem. The HMA bands were generally sharper and more satisfactory on the Phast gels than on conventional polyacrylamide gels and the use of Phast gels was an improvement over conventional PAGE. Non-B subtype viruses could be distinguished from B subtypes, but it was more difficult to distinguish between the non-B subtypes and to assign a subtype to them. Thus, HMA can be adapted to offer a rapid screening method for HIV-1 subtyping, but sequencing is still necessary to assign a definitive subtype. This reflects the empirical nature of the subtype definitions and the quasispecies nature of the HIV genome population.
Novitsky
V
V
Molecular Biology Unit, Central Public Health Laboratory, London, UK.
Arnold
C
C
Clewley
J P
JP
D004279:DNA, Viral / Q000032:analysis*; D005240:Feasibility Studies; D015658:HIV Infections / Q000473:pathology / Q000821:virology*; D015497:HIV-1 / Q000145:classification* / Q000235:genetics*; D006801:Humans; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis*; D010802:Phylogeny; D015203:Reproducibility of Results
D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes