215
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
Glossary
Advanced Setup
In the 7500 software, a feature that allows you to set up your experiment according to your
experiment design. Advanced Setup provides you with maximum flexibility in the design
and setup of your experiment.
AIF
See assay information file (AIF).
allele
For a given target, any of the different sequences that occurs in the population.
allelic
discrimination plot
Display of data collected during the post-PCR read. The allelic discrimination plot is a
graph of the normalized reporter signal from the allele 1 probe plotted against the
normalized reporter signal from the allele 2 probe.
amplicon
A segment of DNA amplified during PCR.
amplification
Part of the instrument run in which PCR produces amplification of the target. For
quantitation experiments, fluorescence data collected during amplification are displayed in
an amplification plot, and the data are used to calculate results. For genotyping or
presence/absence experiments, fluorescence data collected during amplification are
displayed in an amplification plot, and the data can be used for troubleshooting.
amplification
efficiency (EFF%)
Calculation of efficiency of the PCR amplification. The amplification efficiency is
calculated using the slope of the regression line in the standard curve. A slope close to
−
3.32 indicates optimal, 100% PCR amplification efficiency. Factors that affect
amplification efficiency:
•
Range of standard quantities
– To increase the accuracy and precision of the
efficiency measurement, use a broad range of standard quantities, 5 to 6 logs
(10
5
to 10
6
fold).
•
Number of standard replicates
– To increase the precision of the standard quantities
and decrease the effects of pipetting inaccuracies, include replicates.
•
PCR inhibitors
– PCR inhibitors in the reaction can reduce amplification and alter
measurements of the efficiency.
amplification plot
Display of data collected during the cycling stage of PCR amplification. Can be viewed as:
• Baseline-corrected normalized reporter (
∆
Rn) vs. cycle
• Normalized reporter (Rn) vs. cycle
• Threshold cycle (C
T
) vs. well
