Real-Time Fluorescence Detection of a Single DNA Molecule

The fluorescence detection of polymerase chain reaction (PCR) products using the ABI Model 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA, USA) allows researchers to rapidly detect and quantify gene sequences without the need for labor-intensive post-PCR processing such as gel electrophoresis and radioactive hybridization (1). This, in conjunction with a built-in, 96-well format thermal cycler, can greatly increase the throughput of PCR testing. The method uses a fluorescent oligonucleotide probe with a 5′ reporter dye and a downstream, 3′ quencher dye (2). During PCR, the 5′→3′ nuclease activity of Taq DNA polymerase releases the reporter, whose fluorescence can then be detected by the laser detector of the Model 7700 system. The entire process takes approximately 3 h from DNA amplification to data collection, whereas conventional detection methods such as Southern blotting and radioactive hybridization may require several days. Because both amplification and detection processes are contained within a closed system, it minimizes the potential for product carry-over contamination. Initial reports have demonstrated that high levels of DNA target (>100 copies) in a relatively low DNA background (<1 μg) can be detected using this system. We describe a method for the real-time detection of a single-copy Herpes simplex thymidine kinase gene in a high background of genomic DNA that is equivalent to 500 000 diploid mammalian cells (3.3 μg). A 417-bp sequence was amplified and detected using the primer set 5′CGAGACAATCGCGAACATCTAC-3′ (forward) and 5′-GCCAGCATAGCCAGGTCAAG-3′ (reverse) and the probe 5′-CCGGCACAAACATCGTGT-3′. The probe was covalently linked with the reporter dye FAM at the 5′ end and the quencher dye TAMRA at the 3′ end. The PCR amplification was performed using a 96-well optical tray and caps (MicroAmp; Perkin-Elmer, Norwalk, CT, USA). The final reaction mixture of 100 μL consisted of 300 nM each primer, 200 nM probe, 5 U AmpliTaq Gold (Perkin-Elmer), 200 μM each dATP, dCTP and dGTP, 400 μM dUTP, 5.5 mM MgCl2, 8% glycerol, 1 U AmpErase uracil N-glycosylase (UNG) and 1× TaqMan Buffer A containing a reference dye, ROX (all reagents from Perkin-Elmer). The amplification profile was as follows: 50°C for 2 min (enzymatic degradation of carry-over DNA by UNG), 95°C for 10 min (hot start), followed by 60 cycles at 95°C for 15 s and 60°C for 1.5 min. To determine the limit of detection, standards were prepared using 3.3 μg of genomic DNA extracted from human whole blood spiked with 0, 1 and 10 copies of genomic DNA from cell line PA317/ G1Tk1SvNa.7. A no-template control was also included. Cell line PA317/G1Tk1SvNa.7 was cloned from