Snap uv free gel purification kit

Plasma samples were used to generate Envelope-expressing plasmids. Viral RNA was isolated (QIAamp Viral RNA kit, QIAgen, Spain) and a fragment corresponding to the rev, vpu and env genes was amplified using the NLEcoRIF and NLXhoIR primers (nucleotides 5284–5310 and 9055–9027 in the HIV _HXB2 numbering system, respectively) and the RNA-NestedF and the RNA-NestedR primers in a nested PCR (nucleotides 5954–5983 and 8904–8882 in the HIV _HXB2 numbering system, respectively). The PCR fragment was purified (SNAP UV-free gel purification kit, Invitrogen) and subsequently, directionally cloned into the expression vector pcDNA.3.1D/V5/His-TOPO (Invitrogen). Between 10 and 15 recombinant expression plasmids were obtained from each patient and the envelope region was fully sequenced using specific primers with the Big Dye Terminator v3.1 cycle sequencing kit and the ABI 3100 sequence analyzer (Applied Biosystems, Foster City, California, USA). All sequences were aligned and edited using the programs Sequencher v4.7 (Gene Codes Corporation, Ann Arbor, MI) and GeneDoc v2.6. Full-length envelope clones were classified and used depending on their mutations. The regions corresponding to the first and the second exons of Rev were also sequenced in some of the plasmids.

Inverse PCR, semi-specific PCR, as well as GenomeWalker™ analysis were employed to investigate the insertions at the deletion breakpoints of patients DA-77 and ASB4-55. Inverse PCR, as schematically described in Figure S25 (Additional file 1), was performed with the restriction enzymes and PCR primers listed in Tables S28 and S29 (Additional file 1). Semi-specific PCRs, employed according to the principle described in Figure S26 (Additional file 1), were performed with primers summarized in Tables S30 and S31 (Additional file 1). PCR products obtained from these assays were then investigated by sequence analysis. To perform GenomeWalker™ analysis (Clontech, Saint-Germain-en-Laye, France), genomic DNA (2.5 μg per experiment) was restriction digested and adaptors were ligated to the DNA fragments. Subsequently, PCR was performed with an adaptor-specific primer in combination with a primer located close to the breakpoint regions (Figure S27 in Additional file 1). PCRs were performed with the Advantage® 2 PCR Kit (Clontech). The corresponding PCR products were gel-purified (S.N.A.P.™ UV-Free Gel Purification Kit, Invitrogen, USA) and cloned for sequence analyses. The restriction enzymes used for each experiment, together with the region-specific primers, are listed in Tables S32 and S33 (Additional file 1).