Platinum taq dna polymerase enzyme

Genomic DNA was extracted from whole blood samples (QIAamp® DNA Blood Mini Kit/QIAGEN, Qiagen, Hilden, Germany). The complete HIV-1 genome was amplified by nested-PCR employing Platinum Taq DNA polymerase enzyme (Invitrogen, Carlsbad, CA) into four overlapping fragments using HIV-1 specific primers, as following: fragment 1- SCAOSD/LR51 external primers and SCANSD/DP11 internal primers (408–2594), fragment 2- DP10/SCCNAS external primers and DP16/SCCOAS internal primers (2253–4830); fragment 3- MMINT8/ED14 external primers and MMINT3/ED12 internal primers (4653–7811); fragment 4- ED5/SCDOAD external primers and JH44/LTR2 internal primers (6954–9625) (S2 Table) [38 (link)–40 (link)], all positions were relative to HXB2 genome. Isolates with all four fragments completely sequenced were considered full length genomes (FLG); isolates with three complete fragments were considered near full length genomes (NFLG), and isolates with one or two fragment sequences were referred as partial genomes.

HAdV-positive samples obtained by qPCR were subjected to conventional PCR targeting a conserved region of the hexon gene using the primer pair hex1deg and hex2deg^{66 (link)}. The reactions were performed using the Platinum Taq DNA Polymerase enzyme (Invitrogen), following the manufacturer’s recommendations. The expected amplicons of 301 base pairs (bp) were purified using the QIAquick Gel Extraction Kit (Qiagen) following the manufacturer’s recommendation. Sequencing reactions of the purified amplicons were performed using the Big Dye Terminator v. 3.1 Cycle Sequencing Ready Reaction Kit on an ABI Prism 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) at the Fiocruz Institutional Genomic Platform for DNA sequencing (PDTIS).