Methods for viral RNA isolation and PCR amplification for sequencing of near full-length HIV-1 genomes

Kimdar Sherefa Kemal, Milan Reinis, Barbara Weiser, Harold Burger

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

Abstract

HIV-1 in plasma represents the viral quasispecies replicating in the patient at any given time. Studies of HIV-1 viral RNA from plasma or other body fluids therefore reflect the virus present in real time. To obtain near full-length genomic sequences derived from virion RNA it is first necessary to carefully isolate and amplify the RNA. The procedure described below, involves viral RNA extraction, reverse transcription (RT) of the extracted RNA to produce cDNA copies, and PCR amplification of long HIV-1 gene fragments using site-specific, overlapping primers. The primers are based on subtype B HIV-1 strains, and plasma specimens are used in the procedures. However, the protocol can easily be adapted to other HIV-1 subtypes by modifying the primers to match the subtype of interest.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
Pages3-14
Number of pages12
Volume485
DOIs
StatePublished - 2009
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume485
ISSN (Print)10643745

Keywords

  • HIV-1
  • HIV-1 primers
  • HIV-1 viral RNA
  • Long RT-PCR amplification of HIV-1

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Kemal, K. S., Reinis, M., Weiser, B., & Burger, H. (2009). Methods for viral RNA isolation and PCR amplification for sequencing of near full-length HIV-1 genomes. In Methods in Molecular Biology (Vol. 485, pp. 3-14). (Methods in Molecular Biology; Vol. 485). https://doi.org/10.1007/978-1-59745-170-3_1