Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

Appendix DTheory of OperationReal-Time Data AnalysisKinetic Analysis/Quantitative PCRThe 7900HT instrument can be used to determine the absolute or relative quantity ofa target nucleic acid sequence in a test sample by analyzing the cycle-to-cycle changein fluorescence signal as a result of amplification during a PCR. This form ofquantitative PCR analysis, called “kinetic analysis,” was first described using anon-sequence-specific fluorescent dye, ethidium bromide, to detect PCR product(Higuchi et al., 1992; Higuchi et al., 1993). The use of TaqMan probes and reagentsfurther enhances the method by providing sequence-specific amplification ofmultiple targets for ‘comparative’ or ‘relative’ quantification. The fewer cycles ittakes to reach a detectable level of fluorescence, the greater the initial copy numberof the target nucleic acid.Amplification Plot8.02.01.0Phase 3, plateauPhase 2, linearPhase 1, geometricR n0.0010203040Cycle NumberFigure D-3Phases of a Typical Amplification CurveWhen graphed in real-time on a linear scale, normal amplification of PCR productgenerates a curve similar to the one shown in Figure D-3. This ‘amplification’ curveconsists of three distinct regions that characterize the progression of the PCR.Phase 1: Geometric (Exponential)Detection of the high-precision geometric phase is the key to high-precisionquantitative PCR. The geometric phase is a cycle range of high precision duringwhich amplification is characterized by a high and constant efficiency. It occursbetween the first detectable rise in fluorescence and before the beginning of thelinear phase. When plotted on a log scale of DNA vs. cycle number, the curvegenerated by the geometric phase should approximate a straight line with a slope.The 7900HT instrument typically delivers sufficient sensitivity to detect at least3 cycles in the geometric phase, assuming reasonably optimized PCR conditions.D-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_Theory.fm