Synthesis of Radioactive Single-Stranded DNA Probes Using Asymmetrical PCR and Oligonucleotide Random Priming

Single-stranded (ss)DNA labeled probes can be used in S1 nuclease analysis, Southern, northern blotting and in situ hybridization (1,4,7,8). They are especially useful in detecting the specific expression of genes in which both strands are transcribed. Several methods for synthesis of radioactive ssDNA probes have been described (3,6,9), for example, cloning the sequence into a suitable vector like the single-strand bacteriophage M13 for ssDNA production. Asymmetrical polymerase chain reaction (PCR), in which a large excess in the amount of one primer to the other, can also be used to produce an ssDNA for use as a probe. Other investigators using asymmetrical PCR have incorporated 32P into the PCR as 32P-labeled nucleotides (3,9) or as the 5′ 32P-labeled primers to directly radiolabel the probe during synthesis (8). Thus, all subsequent steps must be carried out under precautions for radioactive materials, and the probe must be used before decay of the 32P. In addition, if a 5′ 32P-labeled primer is used, the specific activity of the radioactive probe will be limited to that of the 5′ 32P-labeled primers. Unidirectional PCR has also been used to generate ssDNA probes (3,9). However, this approach requires a much larger amount of template. Here, we introduce a method that combines asymmetrical PCR and oligonucleotide random priming in separate steps to produce radioactive ssDNA probes for use in Northern blotting. We studied the expression of the UL34.5 gene of herpes simplex virus type-1 (HSV-1) grown in tissue culture cells. Both strands of the viral DNA are transcribed in the UL 34.5 gene locus (5). A previously developed PCR protocol (2) was used to produce ssDNA probes. The template for the PCR was a pBR322 plasmid (2.0 μg) containing the EcoRI EK fragment of HSV-1 KOS strain and the UL34.5 gene. Viral DNA could also be used as template. Due to the high GC content of the gene and the failure of several other approaches, PCR was performed using the MasterAmp PCR Optimization Kit and Tfl DNA Polymerase (both from Epicentre Technologies, Madison, WI, USA). Primers were chosen based on their unique sequences and to bracket the UL34.5 gene (2). The first step was to determine the appropriate ratio of primers (data not shown). The best ratio of primer pairs was 50:1 (50 pmol vs. 1 pmol in 50-μL reaction volume) producing the largest amount of ssDNA (Figure 1A). The reaction mixture contained 50 mM TrisHCl (pH 9.0, 25°C), 20 mM (NH4)2SO4 and 200 μM each dNTP, 2.5 mM MgCl2, 4× MasterAmp PCR Enhancer (Epicentre Technologies) and 1.0 U Tfl DNA polymerase. The program consisted of 5 min at 96°C, 35 cycles of 1 min each at 95°C, 60°C and 68°C with an additional incubation of 10 min at 68°C. For comparison, the purified normal PCR product, with 800 bp doublestranded (ds)DNA, was denatured by heat or alkali to produce ssDNA and electrophoresed on a 1.2% agarose gel with the asymmetrical PCR products. The asymmetrical PCR produced two products; the predominant DNA product had the same mobility as the ssDNA produced by denaturation of the 800 bp dsDNA identifying it as ssDNA (Figure 1B). After electrophoresis on a lowermelting gel, the ssDNA band from the asymmetrical PCR was excised, phenolextracted, and its concentration was measured by UV absorption. The amount of ssDNA ranged from 30–90 ng/μL in comparison with 50 ng/μL of normal PCR dsDNA product. The purified ssDNA was stored and used in later labeling reactions. Using DECA prime II from Ambion (Austin, TX, USA) in a random priming reaction, 25–30 ng of ssDNA from either strand was mixed with 2.5 μL 10× primer. After adding nucleasefree H2O to 14 μL, it was boiled for 10 min and chilled on ice. The Klenow polymerase (1.0 μL), buffer (5.0 μL, 5×) and 32P-CTP (5.0 μL) (10 mCi/mL; ICN Biomedicals, Costa Mesa, CA, USA) were added together and incubated for 1 h at room temperature. The radioactive probe was then purified from unincorporated nucleotides using a Sephadex G-50 column (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The specific activity of the probe was approximately 2 × 109 counts per minute (cpm)/μg. The specificity of the desired DNA strand was checked using a dot-blotting method. dsDNA from normal PCR of UL34.5 and ssDNA from either strand of the UL34.5 gene region, produced by asymmetrical PCR, were blotted as 100, 10 and 1 pg onto nitrocellulose membranes (NitroPure; Osmonics, Minnetonka, MN, USA). After denaturation and neutralization, the membranes were hybridized with 32P-laBenchmarks