A new non-fluorescent quencher for use in oligonucleotide synthesis
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Transcript of A new non-fluorescent quencher for use in oligonucleotide synthesis
IntroductionOligonucleotide probes used for qPCR are typically labelled with
a fluorescent dye to enable detection of the target sequence. In
most cases the probe is a doubly labelled oligo where one dye acts
as a fluorophore and the other as a quencher. In order to increase
the sensitivity of the PCR assay, it is desirable for the quencher to
have no native fluorescence (dark quencher). To ease preparation
of probes for use in assays with different fluorophores it would be
advantageous to have a quencher capable of quenching a range of
fluorophores; to this end we have developed a new non-fluorescent
quencher with a broad absorption range.
A series of double-dye probes have been synthesised to evaluate
the efficiency of a new non-fluorescent quencher when paired
with FAM-C7, Cy3™, Cy5™ and Cy5.5™ and for use in RT-PCR. The
quencher was evaluated when incorporated at the 5’ end of the
oligonucleotide.
The new quencher was compared to existing commercially
available quenchers such as Deep Dark Quencher-1 and Black Hole
Quenchers™.
Experimental
Conditions for Taqman™ Assay
Experiments were carried out with the Taqman™ probe and no
target as a negative control. All reactions were carried out in
duplicate.
Sample reaction conditions:
• Reaction in 10 μL volume
• 0.2 mM dNTP
• 0.5 μM primers
• 0.25 μM probe
• 3.0 mM MgCl2
• 0.5U HotStar (Qiagen) Taq
• 10 fmol of target
Thermal protocol:
• Activation of the enzyme: 95°C/15min
• Cycles (40 cycle): 95°C/5s, 55°C/30s, 70°C/30s
• Record at 55°C in each cycle
Results & DiscussionFigures 2-5 show the results of the ‘Link’-quencher compared to
BHQ-1™ and BHQ-2™ at the 5’-end paired with FAM-C7, Cy3, Cy5 and
Cy5.5 at the 3’-end. The graphs show the fluorescence intensity of
the Taqman™ probes against number of cycles.
In the PCR reaction with FAM as the fluorophore, the ‘Link’-
quencher shows some activity as a quencher of FAM, however is
not as efficient as BHQ-1™ and BHQ-2™ (Figure 2).
When the ‘Link’-quencher is used in combination with Cy3™ as the
fluorophore it performs much more efficiently and is comparable
to BHQ-1™ and BHQ-2™ (Figure 3).
With Cy5™, the quencher showed moderate activity but was
significantly less active than BHQ-2™. BHQ-1™ also showed moderate
activity (Figure 4).
When the ‘Link’-quencher was used in conjunction with Cy5.5™ it
showed no activity as a quencher (Figure 5). This was expected, as
there was only a small overlap between ‘Link’-quencher and Cy5.5™
in the UV spectra (see Figure 1).
ConclusionsFrom the data it can be seen that the ‘Link’-quencher works best
when paired with Cy3™ and has some activity as a quencher with
FAM-C7 and Cy5™. Due to the poor overlap with Cy5.5™, virtually
no activity was seen with this pairing. Some impurities were carried
through with the oligos and the quenchers were at the atypical 5’
end. Work is currently under way to repeat these experiments with
the quencher at the 3’ end and the results will be available soon.
Although successful for Cy3™ the ‘Link’-quencher did not perform
over as wide a range as anticipated but it does fill a gap between
DDQ-1 and DDQ-2. We are currently evaluating other molecules for
use as a long-range quencher.
Further InformationFor further information please contact Dr Catherine McKeen,
Technical Manager, Link Technologies ([email protected])
or Dr Grant McGeoch, NPI Team Leader, Link Technologies Ltd
Cy3™, Cy5™ and Cy5.5™ are trademarks of GE Healthcare. Black
Hole Quencher, BHQ-1 and BHQ-2 are trademarks of Biosearch
Technologies, Inc.
www.linktech.co.uk
A new non-fluorescent quencher for use in oligonucleotide synthesisSheena Aitken, Jennifer Mathieson, Grant McGeoch and Catherine McKeen; Link Technologies Ltd, Bellshill, UK.
Tom Brown and Nittaya Gale; ATD Bio Ltd, Southampton, UK.
Figure 1. FAM C7: Excitation at 450-490nm, Emission at 510-530 nm. Figure 2. Cy3™: Excitation at 515-535 nm, Emission at 560-580 nm.
Figure 3. Cy5™: Excitation at 620-650 nm, Emission at 675-690 nm. Figure 4. Cy5.5™: Excitation at 620-650 nm, Emission at 675-690 nm.Figure 1. UV spectrum of ‘Link’-quencher in an oligo.
400 500 600300 700nm