Real-time quantitative PCR is a novel method for accurately and reproducibly quantifying gene copies. It uses a dual-labeled fluorogenic probe (TaqMan Probe) to measure PCR product accumulation in real time, eliminating the need for post-PCR sample handling and reducing contamination risks. This method offers a large dynamic range (at least five orders of magnitude) and is more accurate and less labor-intensive than traditional quantitative PCR methods. Quantitative nucleic acid sequence analysis is crucial in biological research, including gene expression monitoring and genome quantity determination. Real-time PCR has proven to be a powerful tool for quantitative nucleic acid analysis, enabling the analysis of minimal starting quantities of nucleic acid. However, proper design of controls is essential for accurate quantitation. Several methods have been developed for quantitative PCR and RT-PCR, including measuring PCR product quantity during the log phase of the reaction and using quantitative competitive PCR (QC-PCR) with an internal control competitor. QC-PCR is easier to use as it does not require analysis during the log phase. Various detection systems are used for quantitative PCR and RT-PCR, including agarose gels, fluorescent labeling with laser-induced fluorescence, and plate capture hybridization. However, these methods require post-PCR manipulations, which can lead to contamination and limit sample throughput. The authors developed a novel assay for quantitative DNA analysis based on the 5' nuclease assay. This method uses the 5' nuclease activity of Taq polymerase to cleave a nonextensible hybridization probe during the extension phase of PCR. The method uses dual-labeled fluorogenic hybridization probes, with one fluorescent dye serving as a reporter and the other as a quencher. The nuclease degradation of the hybridization probe releases the quenching of the reporter dye, resulting in an increase in fluorescent emission. The method was tested using a plasmid-encoding human factor VIII gene sequence as a model therapeutic gene. The assay uses fluorescent TaqMan methodology and an instrument capable of measuring fluorescence in real time. The method was validated by analyzing 10 replicate sample preparations and showed high reproducibility. The method was also used to quantify a plasmid after transient transfection, demonstrating its ability to accurately measure the quantity of input target sequences. The results showed that the quantity of factor VIII plasmid associated with 293 cells decreased with decreasing plasmid concentration used in the transfection. Real-time PCR offers several advantages over other methods, including a closed-tube system that reduces contamination, the ability to use normalization genes, and the capacity for high-throughput analysis. The method is highly reproducible and allows for a large dynamic range of starting target quantities. It is also compatible with automation technology and can be used for various applications, including quantitative gene expression, gene copy assays, genotyping, and immunoPCR.Real-time quantitative PCR is a novel method for accurately and reproducibly quantifying gene copies. It uses a dual-labeled fluorogenic probe (TaqMan Probe) to measure PCR product accumulation in real time, eliminating the need for post-PCR sample handling and reducing contamination risks. This method offers a large dynamic range (at least five orders of magnitude) and is more accurate and less labor-intensive than traditional quantitative PCR methods. Quantitative nucleic acid sequence analysis is crucial in biological research, including gene expression monitoring and genome quantity determination. Real-time PCR has proven to be a powerful tool for quantitative nucleic acid analysis, enabling the analysis of minimal starting quantities of nucleic acid. However, proper design of controls is essential for accurate quantitation. Several methods have been developed for quantitative PCR and RT-PCR, including measuring PCR product quantity during the log phase of the reaction and using quantitative competitive PCR (QC-PCR) with an internal control competitor. QC-PCR is easier to use as it does not require analysis during the log phase. Various detection systems are used for quantitative PCR and RT-PCR, including agarose gels, fluorescent labeling with laser-induced fluorescence, and plate capture hybridization. However, these methods require post-PCR manipulations, which can lead to contamination and limit sample throughput. The authors developed a novel assay for quantitative DNA analysis based on the 5' nuclease assay. This method uses the 5' nuclease activity of Taq polymerase to cleave a nonextensible hybridization probe during the extension phase of PCR. The method uses dual-labeled fluorogenic hybridization probes, with one fluorescent dye serving as a reporter and the other as a quencher. The nuclease degradation of the hybridization probe releases the quenching of the reporter dye, resulting in an increase in fluorescent emission. The method was tested using a plasmid-encoding human factor VIII gene sequence as a model therapeutic gene. The assay uses fluorescent TaqMan methodology and an instrument capable of measuring fluorescence in real time. The method was validated by analyzing 10 replicate sample preparations and showed high reproducibility. The method was also used to quantify a plasmid after transient transfection, demonstrating its ability to accurately measure the quantity of input target sequences. The results showed that the quantity of factor VIII plasmid associated with 293 cells decreased with decreasing plasmid concentration used in the transfection. Real-time PCR offers several advantages over other methods, including a closed-tube system that reduces contamination, the ability to use normalization genes, and the capacity for high-throughput analysis. The method is highly reproducible and allows for a large dynamic range of starting target quantities. It is also compatible with automation technology and can be used for various applications, including quantitative gene expression, gene copy assays, genotyping, and immunoPCR.