ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

Determining Initial
Template
Concentration and
Cycle Number
A-8 Theory of Operation
Phase 2: Linear
The linear phase is characterized by a leveling effect where the slope of the
amplification curve decreases steadily. At this point, one or more components have
fallen below a critical concentration and the amplification efficiency has begun to
decrease. This phase is termed linear, because amplification approximates an
arithmetic progression, rather than a geometric increase. Because the amplification
efficiency is continually decreasing during the Linear phase, it exhibits low precision.
Phase 3: Plateau
Finally, the amplification curve achieves the plateau phase at which time the PCR
stops and the Rn signal remains relatively constant.
At any given cycle within the geometric phase of PCR, the amount of product is
proportional to the initial number of template copies. When one template is diluted
several times, as with the RNase P target in the TaqMan® RNase P Instrument
Verification Plate (see Appendix D), the ratio of template concentration to detectable
signal is preserved within the exponential phase for all dilutions (see below). This
relationship appears to change as rate of amplification approaches a plateau.
R n
8.0
2.0
1.0
0.0
0
10
20
Cycle Number
30
40
20000
10000
5000
2500
1250