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APPENDIX A – Glossary
Amplicon
– A DNA segment that is amplified during PCR
Amplification Plot
– Displays a plot of the amplification cycles (X axis) versus fluorescence units
(Y axis)
Assay
– Pertaining to the Open qPCR platform, this is a test that contains a PCR master mix and
specific primers/probes to amplify a target sequence
Background Fluorescence
– This represents the baseline of a real-time PCR amplification
curve. This is the fluorescence emitted from free dye, unbound probe, non-specific cleavage of
probe or sample auto-fluorescence
Baseline
– Defined as the PCR cycles in which a reporter fluorescent signal accumulates but is
beneath the instrument’s limit of detection (LOD)
Cq
–
This is the quantification cycle used to calculate starting quantity of the DNA template. A
low Cq value equates to a high quantity of the target sequence in the sample. A high Cq value
equates to a low quantity of the target sequence in the sample.
Cycling Stage
– Also termed as the amplification stage, this refers to a stage that is repeated in
the thermal profile setup.
FAM
– A carboxyfluorescein fluorescent dye with
l
max
(absorption) at 494 nm and
l
max
(emission)
at 518 nm
HEX
– A hexachlorofluorescein fluorescent dye with
l
max
(absorption) at 535 nm and
l
max
(emission) at 556 nm
JOE
– A dechlorinated, dimethoxylated version of the fluorescent dye fluorescein with
l
max
(absorption) at 520 nm and
l
max
(emission) at 548 nm
Melt Curve
– A plot of data that is collected during the melt curve stage. The peaks generated
from a melt curve are indicative of the target melting temperature (T
m
) and can identify
nonspecific PCR amplification
Primer/probe mix
- A component of the PCR reaction containing primers designed for target
amplification and a specific probe designed to detect target amplification
Ramp speed
– This pertains to the speed at which the block temperature changes between
steps during a PCR run
