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 language | English (US) |
|---|---|
| Pages (from-to) | 108-117 |
| Number of pages | 10 |
| Journal | Journal of Virological Methods |
| Volume | 185 |
| Issue number | 1 |
| DOIs | |
| State | Published - Oct 2012 |
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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 journal › Article
}
TY - JOUR
T1 - A novel denaturing heteroduplex tracking assay for genotypic prediction of HIV-1 tropism
AU - Shi, Binshan
AU - Weiser, Barbara
AU - Styer, Linda M.
AU - Kemal, Kimdar
AU - Brunner, Cheryl
AU - Anastos, Kathryn
AU - Burger, Harold
PY - 2012/10
Y1 - 2012/10
N2 - 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.
AB - 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.
KW - Denaturing heteroduplex tracking assay
KW - HIV-1 gp120 V3
KW - Locked Nucleic Acid clamps
KW - Viral tropism
UR - http://www.scopus.com/inward/record.url?scp=84864303846&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84864303846&partnerID=8YFLogxK
U2 - 10.1016/j.jviromet.2012.06.013
DO - 10.1016/j.jviromet.2012.06.013
M3 - Article
C2 - 22728273
AN - SCOPUS:84864303846
VL - 185
SP - 108
EP - 117
JO - Journal of Virological Methods
JF - Journal of Virological Methods
SN - 0166-0934
IS - 1
ER -