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.

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

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 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 microM, 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.

Effect of interaction between noise and toluene on auditory function in the rat.

Rats were exposed to toluene (1000 ppm, 16 h/d, 5 d/w, 2 w), or noise (100 dB Leq, 10 h/d, 7 d/w, 4 w) or toluene followed by noise. Auditory function was tested by brainstem audiometry using a 1/3 octave filtered sine wave stimulus at the frequencies 1.6, 3.15, 6.3, 12.5 and 20.0 kHz. A high-frequency auditory impairment was observed after exposure to toluene alone and noise alone. A slight recovery was recorded 1 and 6 months after the toluene exposure. Toluene followed by noise resulted in a higher threshold at all frequencies. A slight recovery was recorded 6 months post-exposure. The threshold shift exceeded the summated loss caused by toluene alone and by noise alone, particularly at 3.15 and 6.3 kHz. The latencies varied only slightly. The results indicate that the major cause of the auditory impairment was cochlear damage and that only minor injury was caused to the auditory pathways.