Amplification of JCV T-Antigen genome in colon cancer samples.

<p>A schematic representation of the Mad-1 strain of the JCV genomic structure showing viral genes in red, early and late transcripts in grey, and the locations of primers used for T-Antigen amplification (PEP1 and PEP2) and Southern blot probe synthesis (PEP INT1 and PEP INT2) (Panel A). Genomic sequences of the viral early region, which encodes T-Antigen, were detected in 28 of 34 selected samples. A representative Southern blot is presented, with case numbers above each lane. Negative results were observed when the same cases were tested with probes for BKV and SV40. Negative controls show results from reactions containing no DNA and positive control represents gene amplification using pBJC, a plasmid containing JCV DNA as a template or plasmids containing BKV and SV40 T-Antigens. PCR for GAPDH and agarose electrophoresis was used to confirm the presence of intact genomic DNA in all cases. (Panel B). Southern blots of plasmids containing the T-Antigen of JCV, BKV and SV40 were tested with specific probes for each polyomavirus, demonstrating the specificity of the probes (Panel C). A representative Southern blot of PCR amplification of the control regulatory region is shown (Panel D, left). Sequencing revealed the presence of Mad-1 and Mad-4 strains with point mutations in the different samples. A representative sequence is shown in blue, underneath the known sequence of the Mad-4 CR region (black); point mutations are highlighted in red (Panel D, right).