enables target-specific quantification (e.g., gender determination, pathological diagnosis, determination of GMO presence, and species identification).
Real-time PCR template DNA quantification estimates are derived from measured fluorescence accumulation, which is correlated directly with the amount of amplified PCR products produced as the reaction progresses (Heid et al., 1996). Fluorescence is generated either by intercalating dyes that are specific for double-stranded DNA (Wittwer et al., 1997a,b) or by sequence-specific oligonucleotide probes (Holland et al., 1991;Livak et al., 1997). The real-time PCR sequence detection system measures the reporter signal (R) and normalizes it to a passive reference dye. Normalizing accounts for minor well-to-well variations in signal strength, allowing for more accurate sample-to-sample comparisons. The progressive cleavage of the probe at each PCR cycle leads to an increase in normalized reporter signal (R n ) which is proportional to the initial PCR cycles. Reporter fluorescence values are below the baseline detection capabilities of current real-time PCR systems, resulting in stochastic fluctuations in fluorescence (i.e., background fluorescence). To minimize this stochastic effect, normalized reporter signal is subtracted from background noise in the fluorescence signal. Normalized reporter signal minus the background fluorescence signal (DR n ) is then plotted against cycle number (Figure 3.1).
The real-time PCR fluorescence curve generated by the sequence detection system is composed of four distinct phases. When PCR product and reporter signal accumulate beyond background fluorescence levels, the reaction enters the exponential detection phase. At this point the amplification plot crosses a user-defined detection threshold which is set above the background fluorescence noise, preferable at the beginning of the exponential phase. The fractional cycle number at which the reaction crosses the threshold (C t ) is related inversely to the initial template DNA concentration. As PCR products continues to accumulate, the ratio of Taq DNA polymerase to amplified products decreases, resulting in nonexponential accumulation of amplicons. At this point the reaction enters the linear phase. Once PCR product ceases to accumulate due to assay depletion, DR n values remain relatively constant and the reaction enters the plateau phase.
PCR amplification efficiency is the rate at which a PCR amplicon is generated, generally expressed as a percentage. If a particular PCR product doubles in quantity during the geometric phase of its PCR amplification, the PCR assay has 100% efficiency. The slope of a standard curve is commonly used to estimate the PCR amplification efficiency of a real-time PCR. A real-time PCR standard curve is represented graphically as a semi log regression line plot of C t value versus the log of input nucleic acid. A standard curve slope of Γ3.32 indicates a PCR with 100% efficiency (Table 3.1). Slopes that are more negative than Γ3.32 (e.g., Γ3.9) indicate reactions that are less than 100% efficient. Slopes more positive than Γ3.32 (e.g.,Γ2.5) probably indicate poor sample quality or pipetting problems. A 100% efficient PCR will yield a 10-fold increase in amplified products every 3.32 cycles during the exponential phase of amplification (log 10 ΒΌ 3.3219). However, it is not often the case that this value is met exactly.
Calculating amplification efficiencies therefore allows early detection of nonoptimal assay conditions and will facilitate troubleshooting problematic samples prior to