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

Real-Time Data AnalysisPhase 2: LinearThe linear phase is characterized by a leveling effect where the slope of theamplification curve decreases steadily. At this point, one or more components havefallen below a critical concentration and the amplification efficiency has begun todecrease. This phase is termed linear, because amplification approximates anarithmetic progression, rather than a geometric increase. Because the amplificationefficiency is continually decreasing during the Linear phase, it exhibits lowprecision.Phase 3: PlateauFinally, the amplification curve achieves the plateau phase at which time the PCRstops and the R n signal remains relatively constant.DeterminingInitial TemplateConcentrationand CycleNumberAt any given cycle inside the geometric phase of PCR, the amount of product isproportional to the initial number of template copies. When one template is dilutedseveral times, as with the RNase P target in the RNase P Instrument VerificationPlates (see Appendix C), the ratio of template concentration to detectable signal ispreserved in the exponential phase for all dilutions (see Figure D-4). Thisrelationship appears to change as rate of amplification approaches a plateau.8.02.01.02000010000500025001250R n0.0010203040Cycle NumberFigure D-4 Amplification Plot from a Real-Time Run of an RNase P InstrumentVerification PlateApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide D-5DRAFTSeptember 1, 2004 11:39 am, App_Theory.fm