Large-scale amplification, cloning and sequencing of near full-length HIV-1 subtype C genomes.
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.
Rousseau
Christine M
CM
Department of Microbiology, University of Washington, Seattle, WA 98195-8070, USA. cmr@u.washington.edu
Birditt
Brian A
BA
McKay
Angela R
AR
Stoddard
Julia N
JN
Lee
Tsan Chun
TC
McLaughlin
Sherry
S
Moore
Sarah W
SW
Shindo
Nice
N
Learn
Gerald H
GH
Korber
Bette T
BT
Brander
Christian
C
Goulder
Philip J R
PJ
Kiepiela
Photini
P
Walker
Bruce D
BD
Mullins
James I
JI
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
D004279:DNA, Viral; D012367:RNA, Viral