Techne PrimeQ Allelic Discrimination Analysis

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Technical Note T08-005A

Figure 1

:

The

Results Editor

home screen.

Allelic Discrimination Analysis

General Introduction to Data Analysis

The aim of this Technical Note is to explain the principle of allelic discrimination 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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T08-005A: Allelic Discrimination 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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T08-005A: Allelic Discrimination Analysis

Analysis method: Allelic Discrimination

Allelic discrimination is a technique that is able to detect single base pair differences. It is used to discriminate
between genotypes, mutations and polymorphisms within or between samples by comparing the fluorescence
signal obtained using allele-specific, dye-labelled probes. It is 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).

Principle

A single base mismatch within the region complementary to the probe will reduce its stability and therefore the
Tm. The most common technique to use is the hydrolysis probe assay using dual-labelled probes. A multiplex assay
must be run for this type of analysis as two probes, labelled with different reporter dyes are required. One probe
must be 100% specific for one allele and the second probe 100% specific for the mutant allele. During the PCR, the
fluorescent probes anneal specifically to complementary sequences between the forward and reverse primers sites
on the template DNA. During the extension cycle of the PCR, Taq DNA polymerase cleaves the hybridized probe
and due to separation of the reporter dye from the quenchers, an increase in fluorescence is seen.
Thermodynamically it is far more favourable for only the matching probe to bind to the template than the probe
containing the mismatch.

Figure 3: Principle of allelic discrimination
analysis.

Each homozygous genotype will be

detected by a specific probe and show an

increase in fluorescence for that particular

reporter. A heterozygous genotype will
show increased fluorescence with both

reporters. A scatter plot of end point

fluorescence is used to classify and identify

the genotypes.

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 4 below.