TY - JOUR
T1 - PCR amplification, cloning, and construction of HIV-1 infectious molecular clones from virtually full-length HIV-1 genomes.
AU - Ehrenberg, Philip K.
AU - Michael, Nelson L.
PY - 2005
Y1 - 2005
N2 - The development of mixtures of highly processive and high-fidelity thermostable DNA polymerases has enabled the routine recovery of DNA sequences in excess of 25 kb generated by polymerase chain reaction. This powerful tool has been instrumental in the ability to recover virtually full-length HIV-1 proviral DNA as a single, contiguous fragment. Such fragments allow for the clean interpretation of the genomic organization of HIV-1 provirus, as they are not confounded by molecular mosaicism that accrues to overlapping subgenomic amplification strategies. We detail here a robust procedure to produce virtually full-length, single contiguous 9.2-kb HIV-1 amplimers whose full-length infectious potential is reconstituted upon cloning into long terminal repeat-replacement vectors. Large numbers of HIV-1 proviral clones can now be quickly generated and screened to identify the fraction of the viral quasispecies with the highest capacity for viral replication. The methods used to construct long terminal repeat-replacement vectors, amplify HIV-1 provirus, reconstitute full-length provirus, and recover viral stocks will be illustrated using a circulating recombinant form 1 (CRF01_AE, formerly known as subtype E) primary isolate.
AB - The development of mixtures of highly processive and high-fidelity thermostable DNA polymerases has enabled the routine recovery of DNA sequences in excess of 25 kb generated by polymerase chain reaction. This powerful tool has been instrumental in the ability to recover virtually full-length HIV-1 proviral DNA as a single, contiguous fragment. Such fragments allow for the clean interpretation of the genomic organization of HIV-1 provirus, as they are not confounded by molecular mosaicism that accrues to overlapping subgenomic amplification strategies. We detail here a robust procedure to produce virtually full-length, single contiguous 9.2-kb HIV-1 amplimers whose full-length infectious potential is reconstituted upon cloning into long terminal repeat-replacement vectors. Large numbers of HIV-1 proviral clones can now be quickly generated and screened to identify the fraction of the viral quasispecies with the highest capacity for viral replication. The methods used to construct long terminal repeat-replacement vectors, amplify HIV-1 provirus, reconstitute full-length provirus, and recover viral stocks will be illustrated using a circulating recombinant form 1 (CRF01_AE, formerly known as subtype E) primary isolate.
UR - http://www.scopus.com/inward/record.url?scp=24944486471&partnerID=8YFLogxK
U2 - 10.1385/1-59259-907-9:387
DO - 10.1385/1-59259-907-9:387
M3 - Article
C2 - 16061991
AN - SCOPUS:24944486471
SN - 1064-3745
VL - 304
SP - 387
EP - 398
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
ER -