Identification and recovery of minor HIV-1 variants using the heteroduplex tracking assay and biotinylated probes
Open Access
- 23 October 2008
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 36 (22) , e146
- https://doi.org/10.1093/nar/gkn713
Abstract
We describe a method to identify and recover minor human immunodeficiency virus type 1 (HIV-1) sequence variants from a complex population. The original heteroduplex tracking assay (HTA) was modified by incorporating a biotin tag into the probe to allow for direct sequence determination of the query strand. We used this approach to recover sequences from minor HIV-1 variants in the V3 region of the env gene, and to identify minor drug-resistant variants in pro. The biotin-HTA targeting of the V3 region of env allowed us to detect minor V3 variants, of which 45% were classified as CXCR4-using viruses. In addition, the biotin-protease HTA was able to detect mixtures of wild-type sequence and drug-resistance mutations in four subjects that were not detected by bulk sequence analysis. The biotin-HTA is a robust assay that first separates genetic variants then allows direct sequence analysis of major and minor variants.Keywords
This publication has 26 references indexed in Scilit:
- Deciphering Human Immunodeficiency Virus Type 1 Transmission and Early Envelope Diversification by Single-Genome Amplification and SequencingJournal of Virology, 2008
- Simple PCR Assays Improve the Sensitivity of HIV-1 Subtype B Drug Resistance Testing and Allow Linking of Resistance MutationsPLOS ONE, 2007
- Rationale and Uses of a Public HIV Drug‐Resistance DatabaseThe Journal of Infectious Diseases, 2006
- Blinded, Multicenter Comparison of Methods To Detect a Drug-Resistant Mutant of Human Immunodeficiency Virus Type 1 at Low FrequencyJournal of Clinical Microbiology, 2006
- Emergence of CXCR4-Using Human Immunodeficiency Virus Type 1 (HIV-1) Variants in a Minority of HIV-1-Infected Patients following Treatment with the CCR5 Antagonist Maraviroc Is from a Pretreatment CXCR4-Using Virus ReservoirJournal of Virology, 2006
- Sequencing-Based Detection of Low-Frequency Human Immunodeficiency Virus Type 1 Drug-Resistant Mutants by an RNA/DNA Heteroduplex Generator-Tracking AssayJournal of Virology, 2004
- Turnover of env Variable Region 1 and 2 Genotypes in Subjects with Late-Stage Human Immunodeficiency Virus Type 1 InfectionJournal of Virology, 2003
- Randomized Study of Saquinavir with Ritonavir or Nelfinavir Together with Delavirdine, Adefovir, or Both in Human Immunodeficiency Virus–Infected Adults with Virologic Failure on Indinavir: AIDS Clinical Trials Group Study 359The Journal of Infectious Diseases, 2000
- Tracking Changes in HIV-1 Envelope Quasispecies Using DNA Heteroduplex AnalysisMethods, 1997
- HIV Quasispecies and ResamplingScience, 1996