The use of polymerase chain reaction in generation of biotinylated human papillomavirus DNA probes for in situ hybridization

Abstract

The polymerase chain reaction (PCR) was used to produce biotin-labelled human papillomavirus (HPV) 16- and 18-specific DNA probes for in situ hybridization (ISH). PCR was performed by using Amplitaq TM DNA amplification reagent kit according to the manufacturer's instructions, except that dTTP was substituted by different concentrations of biotinylated dUTP (bio-11-UTP). As template DNA, 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. The highest yield was achieved when the concentrations of dTTP and biotinylated dUTP were 150 and 50 μM, respectively. ISH results compatible with those obtained with biotinylated whole genomic HPV DNA probes were demonstrated when primers from E7 and E6 ORF of the HPV-18 genome were used to produce the biotinylated probe by PCR. With HPV-16, several areas of the genome had to be amplified to generate a PCR probe with equal sensitivity as the whole genomic probe. The background staining was always stronger with the PCR probes than with the whole genomic probes. The sensitivity of the PCR probes does not seem to bear a clear-cut correlation with the size or nucleotide content of the probe, but it might rather depend on the three dimensional structure of the probe and the availability of biotin for the detection system by ISH.