title: Large-scale amplification, cloning and sequencing of near full-length HIV-1 subtype C genomes.
abstract: Full-length HIV-1 genome sequencing provides important data needed to address several vaccine design, molecular epidemiologic and pathogenesis questions. A protocol is presented for obtaining near full-length genomes (NFLGs) from subjects infected with HIV-1 subtype C. This protocol was used to amplify NFLGs from 244 of 366 (67%) samples collected at two clinics in Durban, South Africa (SK and PS). Viral load was directly associated with frequency of successful NFLG amplification for both cohorts (PS; p = 0.005 and SK; p < 0.001). Seventeen of 38 initially NFLG-negative SK samples had variation within the PCR primer binding sites, however only 3 of these were successfully re-amplified using re-designed primers homologous to the target viruses. NFLGs were obtained from 7 of 24 PBMC samples processed from subjects whose plasma did not yield a NFLG. Stable plasmid clones were obtained from all 244 NFLG-positive PCR products, and both strands of each genome were sequenced, using a primary set of 46 primers. These methods thus allow the large-scale collection of HIV-1 NFLGs from populations infected primarily with subtype C. The methods are readily adaptable to other HIV-1 subtypes, and provide materials for viral functional analyses and population-based molecular epidemiology studies that include analysis of viral genome chimerization.
authors.affiliation: Department of Microbiology, University of Washington, Seattle, WA 98195-8070, USA. cmr@u.washington.edu
authors.forename: Christine M
authors.initials: CM
authors.lastname: Rousseau
authors.forename: Brian A
authors.initials: BA
authors.lastname: Birditt
authors.forename: Angela R
authors.initials: AR
authors.lastname: McKay
authors.forename: Julia N
authors.initials: JN
authors.lastname: Stoddard
authors.forename: Tsan Chun
authors.initials: TC
authors.lastname: Lee
authors.forename: Sherry
authors.initials: S
authors.lastname: McLaughlin
authors.forename: Sarah W
authors.initials: SW
authors.lastname: Moore
authors.forename: Nice
authors.initials: N
authors.lastname: Shindo
authors.forename: Gerald H
authors.initials: GH
authors.lastname: Learn
authors.forename: Bette T
authors.initials: BT
authors.lastname: Korber
authors.forename: Christian
authors.initials: C
authors.lastname: Brander
authors.forename: Philip J R
authors.initials: PJ
authors.lastname: Goulder
authors.forename: Photini
authors.initials: P
authors.lastname: Kiepiela
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Walker
authors.forename: James I
authors.initials: JI
authors.lastname: Mullins
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral