A novel denaturing heteroduplex tracking assay for genotypic prediction of HIV-1 tropism

Binshan Shi, Barbara Weiser, Linda M. Styer, Kimdar Kemal, Cheryl Brunner, Kathryn Anastos, Harold Burger

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Human immunodeficiency virus type 1 (HIV-1) is characterized by sequence variability. The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 plays a key role in determination of viral coreceptor usage (tropism) and pathogenesis. This report describes a novel denaturing heteroduplex tracking assay (HTA) to analyze the genetic variation of HIV-1 V3 DNA. It improved upon previous non-denaturing HTA approaches to distinguish HIV-1 CCR5 and CXCR4 tropic viruses in mixed populations. The modifications included the use of a single-stranded fluorescent probe based on the consensus V3 sequence of HIV-1 CCR5 tropic viruses, Locked Nucleic Acid (LNA) " clamps" at both ends of heteroduplex DNA, and denaturing gel electrophoresis using Mutation Detection Enhancement (MDE®) as matrix. The analysis demonstrated that the LNA " clamps" increased its melting temperature (Tm) and the thermal stability of heteroduplex DNA. The partially denaturing gel used a defined concentration of formamide, and significantly induced mobility shifts of heteroduplex DNA that was dependent on the number and patterns of DNA mismatches and insertions/deletions. This new technique successfully detected tropisms of 53 HIV-1 V3 clones of known tropism, and was able to separate and detect multiple V3 DNA variants encoding tropisms for CCR5 or CXCR4 in a mixture. The assay had the sensitivity to detect 0.5% minority species. This method may be useful as a research tool for analysis of viral quasispecies and for genotypic prediction of HIV-1 tropism in clinical specimens.

Original languageEnglish (US)
Pages (from-to)108-117
Number of pages10
JournalJournal of Virological Methods
Volume185
Issue number1
DOIs
StatePublished - Oct 2012

Fingerprint

Tropism
HIV-1
Nucleic Acid Heteroduplexes
DNA
Gels
Viruses
Consensus Sequence
Fluorescent Dyes
Freezing
Electrophoresis
Glycoproteins
Clone Cells
Hot Temperature
Mutation
Temperature
Research
Population

Keywords

  • Denaturing heteroduplex tracking assay
  • HIV-1 gp120 V3
  • Locked Nucleic Acid clamps
  • Viral tropism

ASJC Scopus subject areas

  • Virology

Cite this

A novel denaturing heteroduplex tracking assay for genotypic prediction of HIV-1 tropism. / Shi, Binshan; Weiser, Barbara; Styer, Linda M.; Kemal, Kimdar; Brunner, Cheryl; Anastos, Kathryn; Burger, Harold.

In: Journal of Virological Methods, Vol. 185, No. 1, 10.2012, p. 108-117.

Research output: Contribution to journalArticle

Shi, Binshan ; Weiser, Barbara ; Styer, Linda M. ; Kemal, Kimdar ; Brunner, Cheryl ; Anastos, Kathryn ; Burger, Harold. / A novel denaturing heteroduplex tracking assay for genotypic prediction of HIV-1 tropism. In: Journal of Virological Methods. 2012 ; Vol. 185, No. 1. pp. 108-117.
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