Glossary
221
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
passive reference
A dye that produces fluorescence. Because the passive reference signal should be consistent
across all wells, it is used to normalize the reporter dye signal to account for non-PCR
related fluorescence fluctuations caused by minor well-to-well differences in concentrations
or volume. Normalization to the passive reference signal allows for high data precision.
plate layout
An illustration of the grid of wells and assigned content in the reaction plate. In the
7500/7500 Fast system, the grid contains 8 rows and 12 columns.
In the 7500 software, you can use the plate layout as a selection tool to assign well contents,
to view well assignments, and to view results. The plate layout can be printed, included in
a report, exported, and saved as a slide for a presentation.
point
One standard in a standard curve. The standard quantity for each point in the standard curve
is calculated based on the starting quantity and serial factor.
positive control
In genotyping experiments, a DNA sample with a known genotype, homozygous or
heterozygous. In the 7500 software, the task for the SNP assay in wells that contain a sample
with a known genotype.
post-PCR read
Used in genotyping and presence/absence experiments, the part of the instrument run that
occurs after amplification. In genotyping experiments, fluorescence data collected during
the post-PCR read are displayed in the allelic discrimination plot and used to make allele
calls. In presence/absence experiments, fluorescence data collected during the post-PCR
read are displayed in the presence/absence plot and used to make detection calls. Also called
endpoint read.
pre-PCR read
Used in genotyping and presence/absence experiments, the part of the instrument run that
occurs before amplification. The pre-PCR read is optional but recommended. Fluorescence
data collected during the pre-PCR read can be used to normalize fluorescence data collected
during the post-PCR read.
primer mix
PCR reaction component that contains the forward primer and reverse primer designed to
amplify the target.
primer/probe mix
PCR reaction component that contains the primers designed to amplify the target and a
TaqMan
®
probe designed to detect amplification of the target.
pure dye
See custom dye and system dye.
quantitation method
In quantitation experiments, the method used to determine the quantity of target in the
samples. In 7500/7500 Fast systems, there are three types of quantitation methods: standard
curve, relative standard curve, and comparative C
T
(
∆∆
C
T
).
quantity
In quantitation experiments, the amount of target in the samples. Absolute quantity can refer
to copy number, mass, molarity, or viral load. Relative quantity refers to the fold-difference
between normalized quantity of target in the sample and normalized quantity of target in the
reference sample.
quencher
A molecule attached to the 3
′
end of TaqMan
®
probes to prevent the reporter from emitting
fluorescence while the probe is intact. With TaqMan
®
reagents, a nonfluorescent quencher-
minor groove binder (NFQ-MGB) can be used as the quencher. With SYBR
®
Green
reagents, no quencher is used.
