Bacteriophage t7 gene6 exonuclease

The extracted DNA were amplified by nested PCR reactions using PGMY09/PGMY11 and GP5+/GP6+ primers, as previously described by Zubach et al. [27 (link)] and Awua et al. [26 (link)]. The typing of 46 mucosal HPV types were carried out by a multiplex system based on the xMAP® technology, as previously described by Zubach et al. [27 (link)] and reported by Awua et al. [26 (link)]. Briefly, 46 fluorescence sortable microspheres (Luminex Corporation, Austin, TX) were coupled to the 46 specific probes for the HPV types; 6, 11, 13, 16, 18, 26, 30, 31, 32, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85, 86, 87, 89, 90, and 91. The double-stranded second-round PCR products, labelled with biotin, were made single-stranded by digestion with 2 µL of bacteriophage T7 gene6 exonuclease (New England BioLabs, Pickering, ON, Canada) that removed the non-labelled strand after 40 minutes incubation at room temperature. The single-stranded HPV DNA were incubated for hybridization at 60°C for 10 minutes. Streptavidin-phycoerythrin (PE) (Invitrogen) in 1-tetramethyl ammonium chloride (TMAC) (Sigma), was subsequently added and incubated for 5 minutes at 60°C. Genotype specific hybridizations were detected on a Luminex Liquid Chip 200 flow cytometer (Qiagen) using the Luminex IS software (Luminex).

The extracted DNA was amplified by a nested PCR reaction using PGMY09/PGMY11 and GP5+/GP6+ primers as previously described [30 (link)].
The typing of 46 mucosal HPV types was carried out by a multiplex system based on the xMAP® technology as previously described by Zubach et al., [30 (link)]. Briefly, 46 fluorescence sortable microspheres (Luminex Corporation, Austin, TX) were coupled to the 46 specific probes for HPV types 6, 11, 13, 16, 18, 26, 30, 31, 32, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85, 86, 87, 89, 90 and 91.. The double stranded second round PCR products, labelled with biotin, were made single-stranded by digestion with 2 μL of bacteriophage T7 gene6 exonuclease (New England BioLabs, Pickering, ON, Canada) that removed the non-labelled strand after 40 min incubation at room temperature. The single stranded HPV DNA was incubated for hybridization at 60 °C for 10 min and streptavidin-phycoerythrin (PE) (Invitrogen) in 1-tetramethyl ammonium chloride (TMAC) (Sigma), was added and incubated for 5 min at 60 °C. Genotype specific hybridization was detected on a Luminex Liquid Chip 200 flow cytometer (Qiagen) using the Luminex IS software (Luminex).