Summary
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 fulllength 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.
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Acknowledgments
We wish to thank Drs. Mika Salminen, Deborah Birx, Francine McCutchan, and Merlin Robb for efforts to realize these techniques. The views expressed here are the private opinions of the authors and are not to be considered as official or reflecting the views of the US Army or the US Department of Defense. This work was supported in part by Cooperative Agreement No. DAMD17-93-V-3004, between the US Army Medical Research and Materiel Command and the Henry Jackson Foundation for the Advancement of Military Medicine.
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Ehrenberg, P.K., Michael, N.L. (2005). PCR Amplification, Cloning, and Construction of HIV-1 Infectious Molecular Clones From Virtually Full-Length HIV-1 Genomes. In: Zhu, T. (eds) Human Retrovirus Protocols. Methods in Molecular Biology™, vol 304. Humana Press. https://doi.org/10.1385/1-59259-907-9:387
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DOI: https://doi.org/10.1385/1-59259-907-9:387
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