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

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• 作者：Binshan Shi ; Barbara Weiser ; Linda M. Styer ; Kimdar Kemal ; Cheryl Brunner ; Kathryn Anastos ; Harold Burger
• 关键词：HIV-1 gp120 V3 ; Denaturing heteroduplex tracking assay ; Locked Nucleic Acid clamps ; Viral tropism
• 刊名：Journal of Virological Methods
• 出版年：2012
• 出版时间：October, 2012
• 年：2012
• 卷：185
• 期：1
• 页码：108-117
• 全文大小：1002 K

文摘

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 (m>Tm>_m) 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.