A new non-fluorescent quencher for use in oligonucleotide synthesis

Sheena Aitken, Jennifer Mathieson, Grant McGeoch and Catherine McKeen; Link Technologies Ltd, Bellshill, UK. Tom Brown and Nittaya Gale; ATD Bio Ltd, Southampton, UK. grant@linktech.co.uk

Introduction
Oligonucleotide 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.

Results & Discussion

Figures 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 Linkquencher 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).

Conclusions
From 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 Information
For further information please contact Dr Catherine McKeen, Technical Manager, Link Technologies (catherine@linktech.co.uk) or Dr Grant McGeoch, NPI Team Leader, Link Technologies Ltd (grant@linktech.co.uk). Cy3, Cy5 and Cy5.5 are trademarks of GE Healthcare. Black

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.

Hole Quencher, BHQ-1 and BHQ-2 are trademarks of Biosearch Technologies, Inc. www.linktech.co.uk

Sample reaction conditions:

Reaction in 10 L volume 0.2 mM dNTP 0.5 M primers 0.25 M probe 3.0 mM MgCl2 0.5U HotStar (Qiagen) Taq 10 fmol of target

Thermal protocol:
Activation of the enzyme: 95C/15min Cycles (40 cycle): 95C/5s, 55C/30s, 70C/30s Record at 55C in each cycle

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.

500 600 700 nm Figure 1. UV spectrum of Link-quencher in an oligo.

300

400

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.