High-molecular-weight genomic DNA from biopsy and blood samples was isolated by the standard method of proteinase K digestion and phenol chloroform extraction routinely followed in our laboratory.59Detection of HPV was carried out first by using consensus primers located within the conserved L1 region of HPV genome resulting in amplimer of 450 base-pair (bp)60 (forward primer, 5′-GCMCAGGGWCAT AAYAATGG-3′, reverse primer, 5′-CGTCCMARRGGAWACTGATC-3′ where M = A + C, W = A + T, Y = C + T, R = A + G) and later by type-specific primers for high risk types 16 (amplimers size, 217 bp) and 18 (amplimers size, 100 bp) [HPV 16 forward primer, 5′-AAGGCCAACTAAATGTCAC-3′, reverse primer, 5′-CTGCTTTTATACTAACCGG-3′; HPV 18 forward primer, 5′-ACCTTAATG AAAAACCACGA-3′, reverse primer, 5′-CGTCGTTTAGAGTCGTTCCTG-3′], as described earlier.55 Polymerase chain reaction (PCR) was performed using the in-house PCR protocol routinely followed in our laboratory.59 Briefly, the method involved a 25-μL reaction mix containing 100 to 200 ng of DNA, 10 mmol/L of Tris Cl (pH 8.4), 50 mmol/L of KCl, 1.5 mmol/L of MgCl2, 12.5 μmol/L of each deoxynucleoside triphosphate (deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate), 5 pmol of each oligonucleotide primer, and 0.5 U of Taq DNA polymerase (Perkin-Elmer Biosystems; Foster City, CA). The temperature profile used for amplification constituted an initial denaturation at 95°C for 5 min followed by 30 cycles with denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 30 s, which was extended for 4 min in the final cycle. Amplification of β-globin gene (forward primer, 5′-GAAGAGCCAAGGACAGGTAC-3′, reverse primer, 5′-CAACTT CATCCACGTTACACC-3′) with an amplimer of 268 bp served as the internal control (Fig 1
). The oligonucleotide primers were synthesized in an automated DNA synthesizer (Model 381A; Applied Biosystems; Foster City) and were purified with high-performance liquid chromatography.