Methods for viral RNA isolation and PCR amplification for sequencing of near full-length HIV-1 genomes.
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.
Kemal
Kimdar Sherefa
KS
Wadsworth Center, New York State Department of Health, Albany, NY, USA.
Reinis
Milan
M
Weiser
Barbara
B
Burger
Harold
H
D017931:DNA Primers / Q000235:genetics; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D010949:Plasma / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000302:isolation & purification*; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D012367:RNA, Viral