B. Instrumentation
The ABI PRISM 7700 Sequence Detection System is a flexible system designed to take full advantage of the benefits of fluorogenic probe detection. The 7700 system has a built-in thermalcycler and a laser directed via fiber optic cables to each of the 96 sample wells. The fluorescence emission travels back through the cables to a CCD camera detector. Because each well is irradiated sequentially, the dimensions of the CCD array can be used for spectral resolution of the fluorescent light. Because the 7700 instrument detects an entire fluorescence spectrum, the system is capable of distinguishing and quantitating multiple fluorophores in each sample well. The software analyzes the data by first calculating the contribution of each component dye to the experimental spectrum. Each reporter signal is then divided by the fluorescence of an internal reference dye (ROX) in order to normalize for non-PCR related fluorescence fluctuations occurring well-to-well or over time. The use of this internal reference dye, enabled by the ability to distinguish fluorophores, increases the precision of the data obtained with the 7700 system. The fluorescence emissions of SYBR Green I dye and ROX dye are well resolved, so the benefit of using an internal reference dye is obtained for SYBR Green I dye detection of PCR on the 7700 system. The other advantage of distinguishing fluorophores is that probes labeled with different reporter dyes can be used so that more than one PCR target can be detected in a single tube.
The ability to monitor the real-time progress of the PCR completely revolutionizes the way one approaches PCR-based quantification of DNA and RNA. Reactions are characterized by the point in time during cycling when amplification of a PCR product is first detected rather than the amount of PCR product accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. Figure 2 shows a representative amplification plot and defines the terms used in the quantification analysis. An amplification plot is the plot of fluorescence signal versus cycle number. In the initial cycles of PCR, there is little change in fluorescence signal. This defines the baseline for the amplification plot. An increase in fluorescence above the baseline indicates the detection of accumulated PCR product. A fixed fluorescence threshold can be set above the baseline. The parameter CT (threshold cycle) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. A plot of the log of initial target copy number for a set of standards versus CT is a straight line. Quantification of the amount of target in unknown samples is accomplished by measuring CT and using the standard curve to determine starting copy number. The entire process of calculating CT s, preparing a standard curve, and determining starting copy number for unknowns is performed by the software of the 7700 system.
The polymerase chain reaction (PCR) has revolutionized the detection of DNA and RNA. As little as a single copy of a particular sequence can be specifically amplified and detected. Theoretically, there is a quantitative relationship between amount of starting target sequence and amount of PCR product at any given cycle. In practice, though, it is a common experience for replicate reactions to yield different amounts of PCR product. The development of real-time quantitative PCR has eliminated the variability traditionally associated with quantitative PCR, thus allowing the routine and reliable quantification of PCR products.
