SDNA sequence analysis of the HPV-18 E6 region revealed two variations that did not lead to AA changes. The most frequent variation was a C to G transversion at nt287 (n = 27, 48.2 ) (Fig. 1E), with a less frequent A to C transversion occurring at nt551 (n = 9, 16.1 ) (Fig.1 F).2.4 PCRPCR reagents were mixed under a biosafety-hood in a designated room that was free from potential DNA contaminants. Amplification of the integrated HPV genes was performed with primers that were designed according to the HPV-18 reference sequence published in GenBank (accession number NC_001357) (Table 1). The PCR amplification was performed under the following conditions. An initial 5 min denaturation step at 95uC was followed by 35 amplification cycles, with each cycle including a 45 s denaturation step at 94uC, a 45 s annealing step at 55uC, and a 60 s elongation step at 72uC. Amplification was 23727046 complete after a final 10 min elongation step at 72uC. Each 50 ml PCR reaction contained 4 mmol/L MgCl2, 400 mmol/L dNTPs, 3 U Taq DNA polymerase and 4 pmol primers. Amplicons were visualized on 1.0 agarose gels stained with ethidium bromide under UV transillumination (data not shown).3.4 HPV-18 E7 Sequence VariationsDNA sequence analysis of the HPV-18 E7 region revealed two variations, both of which occurred only once and in the same 68181-17-9 site specimen: a C to T transition at nt640, which did not result in an AA change (Fig. 1G), and an A to G transition at nt864, leading to a N92S AA substitution (n = 1, 1.79 ) (Fig. 1H).3.5 HPV-18 L1 Sequence VariationsDNA sequence analysis of the HPV-18 L1 region revealed six variations: a G to A transition at nt5503, leading to a R25Q AA substitution (n = 23, 41.1 ) (Fig. 1I); a C to G transversion at nt5701 with an AA change of P91R (n = 23, 41.1 ) (Fig. 1J); a C to G transversion at nt6460 with an AA change of P344R (n = 24, 42.9 ) (Fig. 1K); a C to G transversion at nt6625 with an AA change of P399R (n = 25, 44.6 ) (Fig. 1L); a C to G transversion at Oltipraz nt6842 with an AA change of P471R (n = 23, 41.1 ) (Fig. 1M); and G to A transition at nt6906 with an AA change of D493N (n = 12, 21.4 ) (Fig. 1N).2.5 Sequencing and Variations AnalysisPCR products were automatically sequenced using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer’s instructions. The sequences were subsequently analyzed by NCBI Blast and DNAMAN version 5.2.2. HPV-18 DNA nucleotide positions were numbered according to the HPV-18 reference sequence (NC_001357). All data were confirmed by repeating PCR amplification and sequence analysis at least twice.3.6 HPV-18 L2 Sequence VariationsDNA sequence analysis of HPV-18 L2 region revealed two variations that did not lead to AA changes: a C to T transition at nt4915 (n = 11, 19.6 ) (Fig. 1O) and a C to A transversion at nt5147 (n = 10, 17.9 ) (Fig. 1P).ResultsIn this study, nucleotide (nt) sequences of E1 (nt 914 to 2887), E2 (nt 2817 to 3914), E4 (nt 3418 to 3684), E5 (nt 3936 to 4157), E6 (nt 105 to 581), E7 (nt 590 to 907), L1 (nt 5430 to 7136) and L2 (nt 4244 to 5632) were determined. Sequence variations were identified in 33 for the 56 specimens assayed (Table 2). In 23 of 56 (41.1 ) specimens, the nucleotide sequences corresponded exactly to the HPV-18 reference sequence (NC_001357).3.7 HPV-18 E4 and E5 Sequence VariationsNo variations were detected in the HPV-18 E4 and E5 regions.DiscussionA study by Angulo et al. [20] highlighted the fact that coinfection with more than.SDNA sequence analysis of the HPV-18 E6 region revealed two variations that did not lead to AA changes. The most frequent variation was a C to G transversion at nt287 (n = 27, 48.2 ) (Fig. 1E), with a less frequent A to C transversion occurring at nt551 (n = 9, 16.1 ) (Fig.1 F).2.4 PCRPCR reagents were mixed under a biosafety-hood in a designated room that was free from potential DNA contaminants. Amplification of the integrated HPV genes was performed with primers that were designed according to the HPV-18 reference sequence published in GenBank (accession number NC_001357) (Table 1). The PCR amplification was performed under the following conditions. An initial 5 min denaturation step at 95uC was followed by 35 amplification cycles, with each cycle including a 45 s denaturation step at 94uC, a 45 s annealing step at 55uC, and a 60 s elongation step at 72uC. Amplification was 23727046 complete after a final 10 min elongation step at 72uC. Each 50 ml PCR reaction contained 4 mmol/L MgCl2, 400 mmol/L dNTPs, 3 U Taq DNA polymerase and 4 pmol primers. Amplicons were visualized on 1.0 agarose gels stained with ethidium bromide under UV transillumination (data not shown).3.4 HPV-18 E7 Sequence VariationsDNA sequence analysis of the HPV-18 E7 region revealed two variations, both of which occurred only once and in the same specimen: a C to T transition at nt640, which did not result in an AA change (Fig. 1G), and an A to G transition at nt864, leading to a N92S AA substitution (n = 1, 1.79 ) (Fig. 1H).3.5 HPV-18 L1 Sequence VariationsDNA sequence analysis of the HPV-18 L1 region revealed six variations: a G to A transition at nt5503, leading to a R25Q AA substitution (n = 23, 41.1 ) (Fig. 1I); a C to G transversion at nt5701 with an AA change of P91R (n = 23, 41.1 ) (Fig. 1J); a C to G transversion at nt6460 with an AA change of P344R (n = 24, 42.9 ) (Fig. 1K); a C to G transversion at nt6625 with an AA change of P399R (n = 25, 44.6 ) (Fig. 1L); a C to G transversion at nt6842 with an AA change of P471R (n = 23, 41.1 ) (Fig. 1M); and G to A transition at nt6906 with an AA change of D493N (n = 12, 21.4 ) (Fig. 1N).2.5 Sequencing and Variations AnalysisPCR products were automatically sequenced using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer’s instructions. The sequences were subsequently analyzed by NCBI Blast and DNAMAN version 5.2.2. HPV-18 DNA nucleotide positions were numbered according to the HPV-18 reference sequence (NC_001357). All data were confirmed by repeating PCR amplification and sequence analysis at least twice.3.6 HPV-18 L2 Sequence VariationsDNA sequence analysis of HPV-18 L2 region revealed two variations that did not lead to AA changes: a C to T transition at nt4915 (n = 11, 19.6 ) (Fig. 1O) and a C to A transversion at nt5147 (n = 10, 17.9 ) (Fig. 1P).ResultsIn this study, nucleotide (nt) sequences of E1 (nt 914 to 2887), E2 (nt 2817 to 3914), E4 (nt 3418 to 3684), E5 (nt 3936 to 4157), E6 (nt 105 to 581), E7 (nt 590 to 907), L1 (nt 5430 to 7136) and L2 (nt 4244 to 5632) were determined. Sequence variations were identified in 33 for the 56 specimens assayed (Table 2). In 23 of 56 (41.1 ) specimens, the nucleotide sequences corresponded exactly to the HPV-18 reference sequence (NC_001357).3.7 HPV-18 E4 and E5 Sequence VariationsNo variations were detected in the HPV-18 E4 and E5 regions.DiscussionA study by Angulo et al. [20] highlighted the fact that coinfection with more than.
