Techne PrimeQ - Manual (Page 150)

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3.17 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.
Conventional approaches to genotyping and mutation have traditionally relied on a three-stage
process: amplification (e.g. PCR), discrimination (e.g. restriction digest) and detection (often
radioactive), which are often time-consuming, complicated and costly. Allelic discrimination on the
PrimeQ real-time PCR system allows these stages to be performed automatically within the
reaction tube, thereby providing an assay which is rapid, reliable and cost-effective in terms of
both reagents and time.
Principle
A single base mismatch within the region complementary to the probe will reduce its stability and
therefore the Tm. The most common assay to use is the hydrolysis probe assay using dual-
labelled probes. In this assay, PCR master mix, primers and fluorogenic probes with different
labels constructed for the two allele sequences (located between the primer sites) are run in a
thermal cycler with the unknown samples. 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. When the allele is placed in the centre of the probe it has the maximum
effect on the probe-template stability. Thermodynamically it is far more favourable for only the
matching probe to bind to the template than the probe containing the mismatch.
3.17.1 Assay requirements
• Need at least two reporter dyes.
• Need at least two cycles in the stage.
• Need a dye defined as PRD for PRD correction tab to be visible.
• Need at least two readings for baseline correction and analysis methods tabs to be shown.
• Positive control (POS) for each allele
3.17.2 Setup
The setup of an allelic discrimination assay in Quansoft follows a similar path to plus-minus
scoring. Options for baseline correction are chosen and then a choice provided as to which
readings to analyse. The fluorescence level of the reporters is then plotted against each other on a
scatter graph.
• In the Analysis Selection box, highlight the stage name for the analysis to be applied, click
Edit and the Analysis Wizard will launch.
• Choose Allelic Discrimination from the drop-down menu in the Analysis Method Selection
box.
• In the Dye Usage box, assign one reporter as Allele 1 and the other reporter as Allele 2. If
the user attempts to proceed with only one dye assigned then a message will be displayed
telling the user that two dyes must be assigned for this analysis method to be performed.
Raw data for
reporter 1 and
2 with or
without PRD
correction
Baseline
correction
(where >1
reading)
Select
reading(s)
for analysis
Plot reporter 1
against
reporter 2
Report Allelic
discrimination
Raw data for
reporter 1 and
2 with or
without PRD
correction
Baseline
correction
(where >1
reading)
Select
reading(s)
for analysis
Plot reporter 1
against
reporter 2
Report Allelic
discrimination