Several HPV-positive biopsies of uterine exocervix from previous studies on cervical biopsies were also used in randomly-selected runs as a posi- tive control. Polymerase Chain Reaction In order to detect HPV DNA sequence in our cases, including tumor and non-tumor autopsy cases, we performed PCR assay using a pair of general HPV primer which could anneal with and amplify target DNA sequence in the L1 open reading frame of many types of HPV especially the types 6b, 11, 16, 18, 31 and 33. The sequence of forward and reverse general primers as well as the size of expected amplification product is shown in table 1. The PCR assay using general HPV primer set was performed with and without internal control. Internal control was a 100 bp sequence of beta globin gene (as an endogenous target), and was amplified and detected in all of cases using a beta globin gene specific primer set. The sequences of forward and reverse beta globin gene primers are shown in table 1. Briefly, PCR was performed in a total volume of 25 µl prepared by adding appropriate amounts of reaction components into a 0.5 ml Eppendorf microtube. The reaction mixture contained 2.5 µl of 10x buffer, 1.5 mM MgCl2, 0.3 mM of each dNTP, 20 pM of each general HPV primer and 5 pM of each beta globin primer, two unit of Taq DNA polymerase and 300 ng of template DNA sample extracted from the cases under study or 10 ng of p HPV (recombinant plasmid) as positive control. A negative control or blank (all reactants minus target DNA) was also used in every run of PCR. The mixture was overlaid with 40 µl of mineral oil and subjected to 40 cycles of PCR amplification Papilloma virus in esophageal squamous cell carcinoma using an Eppendorf thermocycler and a touch down PCR program shown in table 1. At the end of the PCR, tubes were transferred to refrigerator for later use in agarose gel elec- trophoresis, and analysis of the results. We also examined half of tumoral cases in a heteroduplex PCR using both HPV-16 and HPV-18 specific primer sets. Detection and Analysis of the Reaction Products 5 µl of each PCR product was mixed well with 4 µl of loading dye (Bromophenol blue, EDTA, Glycerol) on a clean surface, and the mixtures were then transferred into the wells created within agarose gel. Along with cases under study, both negative and positive controls as well as DNA size marker were loaded onto gel simultaneously for electrophoresis in a tank containing Tris/Acetic acid/ EDTA (TAE) buffer and little amount of ethid- ium bromide as intercalating dye under 70 volts for 2 hours. After electrophoresis, the gel was transferred onto UV transilluminator to visualize PCR products, if there was any positive reaction (figure 2). Results No HPV genomic sequence was detected in any of 92 cases of ESCC. In other words no specific band was seen on agarose gel after electrophoresis except for a sharp band equal to 100 bp represent- ing amplified beta globin gene sequence used as internal control. Similarly, none of 20 autopsy cases showed evidence of HPV infection in PCR assay. Because PCR assay with internal control showed no amplified HPV DNA sequence in any of the cases, second PCR assay using general HPV primer set was performed, but no internal control was included Table 1: The sequence of primer sets for human papilloma virus (HPV) and beta globin gene used in a touch down PCR program General HPV primer set Product size Forward 5-TTT GTT ACT GTG GTA GAT AC 140 bp Reverse 5-GAA AAA TAA ACT GTA AAT CA Beta globin gene specific primer set Product size Forward 5-ACA CAA CTG TGT TCA CTA GC 100 bp Reverse 5-CAA CTT CAT CCA CGT TCA CC PCR Program -Initial denaturation 94 C for 3 min -denaturation at 94C for 30 seconds -16 cycles with -anealing at 65C to 50C for 2 minutes -extension at 72C for 30 seconds -denaturation at 94C for 30 seconds -14 cycles with -anealing at 50C for 1 minutes -extension at 72C for 30 seconds -denaturation at 94C for 30 seconds -10 cycles with -anealing at 49C to 40C for 1 minutes -extension at 72C for 30 seconds -Final extension 72C for 5 minutes Iran J Med Sci June 2012; Vol 37 No 2 129