TY - CHAP
T1 - Methods for viral RNA isolation and PCR amplification for sequencing of near full-length HIV-1 genomes
AU - Kemal, Kimdar Sherefa
AU - Reinis, Milan
AU - Weiser, Barbara
AU - Burger, Harold
PY - 2009
Y1 - 2009
N2 - 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.
AB - 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.
KW - HIV-1
KW - HIV-1 primers
KW - HIV-1 viral RNA
KW - Long RT-PCR amplification of HIV-1
UR - http://www.scopus.com/inward/record.url?scp=60349095623&partnerID=8YFLogxK
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U2 - 10.1007/978-1-59745-170-3_1
DO - 10.1007/978-1-59745-170-3_1
M3 - Chapter
C2 - 19020814
AN - SCOPUS:60349095623
SN - 9781588298591
VL - 485
T3 - Methods in Molecular Biology
SP - 3
EP - 14
BT - Methods in Molecular Biology
ER -