Fig. 2

Construction of 1-LTR_HIV. (A) Structure of pHXB2. The empty boxes flanking the LTRs represent cellular sequences. (B) Removal of an XhoI/XbaI fragment containing nef sequences, 3’-LTR, and cellular sequences from pHXB2 using the restriction enzyme XhoI that cleaves in the nef sequence and XbaI that cleaves at the end of the cellular sequences. (C) PCR amplification of an XhoI/XbaI DNA fragment containing nef sequences and one LTR with the adjacent viral sequence (primer binding site) containing the restriction site NarI. The source material for the PCR was DNA from HXB2 infected Jurkat cells. The primers were designed from unique sequences flanking the 5’- and 3’-LTR of HXB2 (reported in Materials and methods). The PCR product was purified, analyzed on agarose gel, cloned into the pCRII-Topo vector, and transformed into DH5-α competent cells. Ten transformants were isolated and sequenced (Fig. 3). The PCR reaction was carried out using the Invitrogen Platinum Supermix High Fidelity kit. PCR purification was done using the Qiakit PCR purification kit. Agarose gel extraction was done with the Qiagen gel extraction kit. Competent cells and TA ligation kit were obtained from Invitrogen (Waltham, MA) and restriction enzymes from New England Biolabs (Ipswich, MA). (D) The recombinant plasmid was digested with XhoI and XbaI and the isolated insert was T4 ligated with pHXB2ΔXhoI/XbaI to obtain pHXB2 NarI/NarI. (E) Digestion of this construct with NarI releases the linearized 1-LTR_HIV used in this study. Agarose gel extraction and transformation of the competent cells were performed as described above