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Nucleic Acids Research Advance Access published online on October 23, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn713
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Identification and recovery of minor HIV-1 variants using the heteroduplex tracking assay and biotinylated probes

Gretja Schnell1, William L. Ince2 and Ronald Swanstrom1,2,3,4,*

1Department of Microbiology and Immunology, 2Curriculum in Genetics and Molecular Biology, 3Lineberger Comprehensive Cancer Center and 4UNC Center for AIDS Research, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599-7295, USA

To whom correspondence should be addressed. Tel: 919-966-5710; Fax: 919-966-8212; Email: risunc{at}med.unc.edu

Received September 17, 2008. Revised September 29, 2008. Accepted September 30, 2008.

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.


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