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

Real-Time Data Analysis
Kinetic Analysis/
Quantitative PCR
The 7900HT instrument can be used to determine the absolute or relative quantity of a
target nucleic acid sequence in a test sample by analyzing the cycle-to-cycle change
in fluorescence signal as a result of amplification during a PCR. This form of
quantitative PCR analysis, called “kinetic analysis,” was first described using a
non-sequence-specific fluorescent dye, ethidium bromide, to detect PCR product
(Higuchi et al., 1992; Higuchi et al., 1993). The use of TaqMan probes and reagents
further enhances the method by providing sequence-specific amplification of multiple
targets for ‘comparative’ or ‘relative’ quantification. The fewer cycles it takes to reach a
detectable level of fluorescence, the greater the initial copy number of the target
nucleic acid.
R n
8.0
2.0
1.0
0.0
0
10
Amplification Plot
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Cycle Number
Phase 3, plateau
Phase 2, linear
Phase 1, geometric
When graphed in real-time on a linear scale, normal amplification of PCR product
generates a curve similar to the one shown in the figure above. This ‘amplification’
curve consists 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-precision
quantitative PCR. The geometric phase is a cycle range of high precision during which
is characterized by a high and constant amplification efficiency. It occurs between the
first detectable rise in fluorescence and before the beginning of the Linear phase.
When plotted on a log scale of DNA vs. cycle number, the curve generated by the
geometric phase should approximate a straight line with a slope. The 7900HT
instrument typically delivers sufficient sensitivity to detect at least 3 cycles in the
geometric phase, assuming reasonably optimized PCR conditions.
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40
Theory of Operation A-7