Techne PrimeQ Plus-Minus Scoring Analysis

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Technical Note T08-004A

Figure 1

:

The Results Editor

home screen.

Plus-Minus Scoring Analysis

General Introduction to Data Analysis

The aim of this Technical Note is to explain the principle of plus-minus scoring analysis and to guide you through
the experimental set up and analysis of data. It begins with a general introduction to data analysis.

Results Editor

The starting point for data analysis is the Results Editor. Before an analysis method has been set, the Results Editor
home screen will display the plate layout and thermal cycling program. Each stage where readings have been made
will have its own tab showing a graph of raw data.

Each stage where readings have been made will have a separate results
tab enabling analysis of the data for that particular stage.

The Analysis Selection box displays the stage name as assigned in the
program setup. Only those stages that have been assigned with reads are
displayed since stages without reads have no data to analyse. Double-click
on a stage name, or highlight a stage name then click on Edit to launch the
Analysis Wizard Selection screen.

Selecting the Analysis Method

The Analysis Selection box on the Results Editor home screen allows the user to define the method of analysis to
be applied to the readings gathered during the PCR run. Highlighting a stage name and pressing the Edit button will
launch the Analysis Wizard Selection screen and allow an Analysis Method to be assigned for that stage.

Figure 2: The Analysis Wizard Selection screen.

Analysis Method: The drop-down menu lists analysis types appropriate to

the selected stage. These will be dependent on the number of reads and
the number of cycles programmed into the run.
Dye name: The name of the dyes selected in the program setup will be
displayed.
Dye Usage: Assign a Dye Usage from the list in the drop-down menu. There
will be one dye usage box for each read present in the stage.

Cancel: Aborts the procedure and takes the user back to the Results Editor.
Finish: Accepts all the default analysis settings for the analysis method

chosen and closes the Wizard.

Reset defaults: Returns all analysis parameter settings to the defaults.
Back/Next: Allows the user to move between screens in the Analysis

Wizards.

The Analysis Wizards

Once an analysis method has been defined, a series of default settings will automatically analyse the data. The
defaults can be viewed in the Analysis Wizards and edited if required. The intuitive Analysis Wizards explain in
detail the mathematics behind the analysis settings and will lead you through each stage of the analysis setup.
With experience, analysis can be quickly performed using the Analysis Properties displayed in the Parameters (PAR)
box feature found on each data graph.

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T08-004A: Plus-Minus Scoring Analysis

Analysis Method Options

The following analysis options are available in Quansoft:

Baseline

This simple analysis method allows for correction of differences in background fluorescence. It is also incorporated
into many of the other analysis methods.

Quantification

Quantification analysis is used to determine the absolute or relative quantity of a target DNA template in a given
test sample by measuring the cycle-to-cycle change in the fluorescent signal. The fluorescent signal increases
proportionately to the amount of amplified DNA and quantification is performed either by comparison of the
fluorescence of a PCR product of unknown concentration with that of several dilutions of an external standard, or
by comparing the fluorescence of one product relative to another. To be able to make this comparison, the
fluorophore is measured at a point in the amplification where the reaction efficiency can be considered optimal.
This is generally around the cycle at which an increase in fluorescence is first detected.

Dissociation curve

Dissociation curve analysis can add to the information obtained from the PCR. Also known as melting curve
analysis, it measures the temperature at which the DNA strands separate into single strands. This provides a
measurement of the melting temperature or Tm, taken as the point at which 50% of the double stranded DNA
(dsDNA) molecules are dissociated. Using the easy-to-program ‘ramp’ function, PrimeQ will perform a thermal
ramping program that can be used to determine the Tm of the PCR product. This analysis provides the user with
extra confidence in experiments using intercalating dye chemistry for identifying amplification of non-specific
products or contamination.

Plus-minus scoring

This analysis exploits PrimeQ’s fluorescence technology to determine with ease and accuracy the presence or
absence of a PCR product in any given sample. Input data can either be kinetic (where readings are taken
throughout the amplification stage) or end-point (readings taken at the end of the run). The software scores the
samples as positive or negative according to user-defined thresholds.

Allelic discrimination

Users of PrimeQ have the option of this powerful technique capable of detecting single nucleotide differences
(SNPs). It can be used to discriminate between genotypes, mutations and polymorphisms within or between
samples simply by comparing the fluorescence signal obtained using allele-specific, dye-labeled probes.

Multi-read

This is a simple end point analysis method which will report the average fluorescence of a selected number of
readings. It is useful for assays other than PCR, for example fluorescence-based DNA assays, where just the
fluorescence of a sample needs to be measured; in this way PrimeQ can be used as a simple fluorescence plate
reader or fluorimeter.

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T08-004A: Plus-Minus Scoring Analysis

Analysis method: Plus-Minus Scoring

This analysis method is used to record the presence or absence of a PCR product; it is therefore a qualitative rather
than a quantitative analysis method. The input data for the analysis is usually the end-point fluorescence (a few
readings taken at the end of the run – at least two are required). Since it is not strictly necessary to gather
fluorescence data during the amplification cycles, the amplification can be performed in a non-real time thermal
cycler as long as the reaction contains an appropriate fluorescence detection system for the target. The plate can
then be read a few times in PrimeQ to generate the fluorescence data required for the analysis. Note that
consumables appropriate for use in PrimeQ must be used.

Plus-minus assays can be performed with or without an internal positive control (IPC), although its use can prove
helpful to ensure that a failed PCR is not mistaken for a negative result. A no template control (NTC) is used to set
two confidence thresholds above which all unknowns will be scored as positive and below which they will be
scored as negative. Samples between the two threshold values are scored as undetermined. With no user input,
Quansoft will automatically analyse the raw data (or baseline corrected data) to measure the difference in
fluorescence between the unknown samples and NTC.

Setting up and running the experiment

If the entire run is to be performed on PrimeQ, set up the thermal cycling profile in the usual way. You may or may
not wish to collect fluorescence data at each cycle to view the progress of the amplification; however you will need
to use fluorescence data from a few cycles at the end of the run for data analysis. This may be the last few cycles of
the amplification stage, or a separate stage may be programmed for this purpose. Examples of programs are given
in Figure 3 below.

Figure 3: Example programs for plus-minus assays.

(A) A standard two-step program with reads for

both the target and IPC throughout the

amplification stage. Analysis would be performed

on the data from the final few cycles.

(B) As A, but with an additional stage at the end of

the amplification stage with a few reads for the

plus-minus analysis.

(C) No readings are taken during amplification but

an additional stage is included at the end with a few

reads for the plus-minus analysis.

If multiplexing is possible, an IPC can be amplified simultaneously with the target. This will highlight any samples
which fail to amplify due to inhibition of the reaction. The IPC can be a target known to be present in all the test
samples or can be a target “spiked” into the master mix. It is good practice to also include both positive and
negative controls as well as a NTC.

During the run, the real-time collection of data can be monitored on the Run Screen. The plate layout shows the
fluorescence curve on a per-well basis and the temperature profile plot indicates how far the run has progressed.
When the run has completed, results can be viewed in the Results Editor with data from each stage of the run
located under its own tab.