Thermo Scientific Applied Biosystems QuantStudio 7 Pro - Genotyping run; Presence;absence run; Melt curve run

Genotyping run
A genotyping runs detects the single nucleotide polymorphism (SNP) variants of a target nucleic acid
sequence.
Genotyping runs use preformulated TaqMan
™
SNP Genotyping Assays that include the following
components:
•
Two sequence-specific primers for amplification of sequences containing the SNP of interest
•
Two allele-specific TaqMan
™
probes for Allele 1 and Allele 2
1.
The software normalizes the fluorescence of the reporter dyes to the fluorescence of the passive
reference dye in each well.
2.
The software plots the normalized reporter dye signal of each sample well on an Allelic
Discrimination Plot, which contrasts the reporter dye intensities of the allele-specific probes.
3.
The software algorithmically clusters the sample data, and assigns a genotype call to the samples
of each cluster according to its position on the plot.
Presence/absence run
Presence/absence run determines the presence or absence of a target nucleic acid sequence in a
sample.
The software calls the target present or absent based on an algorithmically determined call threshold.
(The call threshold is dierent from the C
t
threshold; the C
t
threshold is not used to make calls.)
Melt curve run
Melt curve runs determines the melting temperature (Tm) of the amplification products of a PCR that
used intercalating dyes.
In the software, melt curve analysis is included in the default run method for any run type that uses
intercalating dyes.
1.
The software plots a melt curve based on the fluorescence of the dye with respect to change in
temperature.
2.
Using the melt curve, the software calculates the melting temperature (T
m
).
Chapter 1 Product information
Types of runs
1
QuantStudio
™
6 Pro Real-Time PCR System and QuantStudio
™
7 Pro Real-Time PCR System User Guide
33