Fig. 3

PCR amplification of an HIV DNA fragment containing the long terminal repeat (LTR) of the 1-LTR episomal DNA used for the construction of 1-LTR_HIV. (A) Gel analysis of the XhoI/NarI fragment of 1-LTR episomal DNA present in Jurkat cells infected with HXB2. A DNA fragment containing a single LTR was obtained by PCR amplification of total DNA extracted from a Jurkat cell line (J-20, > 99% CD4⁺) acutely infected with HXB2, using the primers described in Fig. 2. The PCR reaction was carried out using the Platinum Supermix High Fidelity kit (Invitrogen). The PCR product was purified using the Qiakit PCR purification kit, analyzed by gel electrophoresis, cloned into the pCRII-Topo vector, and transformed into DH5-α competent cells (competent cells and TA ligation kit from Invitrogen). The size of the amplicon (0.9 kb) is as expected. (B) Sequence analysis of the XhoI/NarI 1-LTR circular DNA fragment amplified from infected Jurkat cells and used to construct 1-LTR_HIV. Plasmid DNA was extracted from 10 transformants using the Qiagen Plasmid Purification kit. Each plasmid contains an insert of the expected size (0.9 kb) as judged by agarose gel analysis of the EcoRI digests of the respective DNAs. The amplified NarI/XbaI insert was sequenced. The 5’ and 3’ ends of the sequence of one of the isolated amplicons are shown, aligned with the sequence of HXB2. All nucleotide sequences determined in the course of this investigation were obtained from the NCI Core Facility at NIH