Techne PrimeQ - Manual (Page 109)

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3.10 Choosing an analysis method
Quansoft allows the user to assign an analysis method to any stage of the PCR that has
fluorescent readings. The available analysis methods are:
• None: When no analysis method has been set, the Results Editor will display the plate
layout and a graph of raw fluorescent data.
• Baseline correction: Allows the user to adjust the data for background fluorescence.
• Quantification: Determines the amount of starting DNA:
o
Absolute quantification: Compares the Cqs of unknown samples against those
of known standards plotted against the log of their concentrations on a standard
curve.
o
Relative quantification: An absolute quantification method; this uses two
reporter dyes and two standard curves to compare the concentration of one DNA
template relative to a second template.
o
Relative quantification cycles: Cq values for a calibrator can be used for a
relative quantification between the calibrator and all other samples in the
experiment. No standards are required.
• Dissociation curve: Measures the temperature at which the DNA strands dissociate (i.e.
the melting temperature or Tm). Since the melting temperature is characteristic of the GC
content, length and sequence of a DNA product, this method is a useful tool in product
identification.
• Plus-minus scoring: Determines the presence or absence of a PCR product – input data
can either be kinetic or end-point (readings taken at the end of the run).
• Allelic discrimination: Detects single nucleotide differences – the most common assay to
use is the hydrolysis probe assay using dual-labelled probes for each of the alleles of
interest.
• Multi-read: Combines all the readings from a well and takes an average – useful in end-
point analysis.
The table below provides a point summary of the valid analysis methods in terms of the number of
cycles and the number of filter reads within a program.
Number of cycles
in the stage Number of filter
reads in the stage Valid analysis methods
Two or more One Baseline
Quantification (without relative quantification options)
Multi-read
Plus-minus (without internal positive control)
Ramp read One Dissociation curve
Two or more Two or more Baseline
Quantification (all options)
Multi-read
Plus-minus
Allelic discrimination
Ramp read Two or more Dissociation curve