Polymerase chain reaction for producing biotinylated human papillomavirus DNA probes for in situ hybridization.
Polymerase chain reaction (PCR) was used to produce biotinylated DNA probes for human papillomavirus (HPV) types 16 and 18. The specificity and sensitivity of the probes were tested with in situ hybridization to detect HPV DNA in cervical biopsies or cell lines (CaSki, SiHa, and HeLa). The Gene Amp DNA Amplification kit (Perkin-Elmer Cetus, Norwalk, CT) was used to perform PCR according to the manufacturer's instructions, except that dTTP was substituted by different concentrations of biotinylated dUTP (bio-11-UTP). As the template DNA, the DNA extracted either from CaSki or HeLa cells was used. The reaction mixture was taken through up to 40 cycles of amplification in a Perkin-Elmer Cetus Thermal Cycler (Perkin-Elmer Cetus, Norwalk, CT). The highest yield was achieved when the concentrations of dTTP and biotinylated dUTP were 150 microM and 50 microM, respectively. In situ hybridization results compatible with those obtained with biotinylated or radioactively labelled whole genomic HPV DNA probes were demonstrated when primers from E6, E7, and L1 ORF of the HPV 18 were used to produce the biotinylated probe by PCR. With HPV 16, the positive signals were always weaker with the PCR probe than with the whole genomic probe. Overall, the PCR probes might have a lower sensitivity than the whole genomic probes. The background stain was always stronger with the PCR probes than with the whole genomic probes, especially with HPV 16 probes. There does not seem to be a clear correlation between the sensitivity of PCR probes and the size or nucleotide content of the probe.
Syrjänen
S
S
Department of Pathology, University of Kuopio, Finland.
Andersson
B
B
Juntunen
L
L
Syrjänen
K
K
D001483:Base Sequence; D001710:Biotin; D002460:Cell Line; D015342:DNA Probes / Q000138:chemical synthesis* / Q000235:genetics; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014412:Tumor Virus Infections / Q000175:diagnosis*; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis
D015342:DNA Probes; D001710:Biotin