3130 genetic analyzer automated sequencer

HPV DNA detection in all FFPE samples (114 samples) was carried out by nested-PCR using MY09/MY11 primer pair (outer primers) and GP5 + /GP6 + primer pair (inner primers) to obtain almost a 150 bp fragment of HPV L1 gene [22 (link)]. The PCR reactions was performed in a 50 μL reaction mixture including 100–200 ng of DNA template, 2.5 mM of MgCl₂, 10 pmol of each primer, 50 mM of each dNTP, and 2 U of Taq DNA polymerase. PCR amplification cycles were as follow for the first and the second rounds: an initial 3-min denaturation at 95 °C, followed by 35 cycles of 95 °C for 30 s, 50 °C for 45 s, and 72 °C for 1 min (first round) and 35 cycles of 95 °C for 30 s, 52 °C for 40 s and 72 °C for 45 s (second round), and a final elongation for 5 min at 72 °C. A reaction mixture lack of DNA template, as a negative control, was included in each run of PCR. The PCR products were run on a 2% agarose gel.
The PCR amplification products were sequenced using BigDye® Terminator v3.1 Cycle Sequencing Kit and a 3130 Genetic Analyzer Automated Sequencer as specified by Applied Biosystems manuals (Foster City, CA). Nucleotide sequences were edited with Bioedit software and converted to FASTA format. Then, sequences compared to other HPV types using the Blast server (http://www.ncbi.nlm.nih.gov/blast/).