Nucleic Acids Research Advance Access originally published online on January 26, 2007
Nucleic Acids Research 2007 35(5):e30; doi:10.1093/nar/gkl1136
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Nucleic Acids Research, 2007, Vol. 35, No. 5 e30
© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Novel DNA probes with low background and high hybridization-triggered fluorescence
Nanogen, Inc., 21720 23rd Drive SE, Suite 150, Bothell, WA 98021, USA
*To whom correspondence should be addressed. Tel: +1 425 482 5168; Fax: +1 425 482 5550; E-mail: elukhtanov{at}nanogen.com
Received October 13, 2006. Revised December 8, 2006. Accepted December 13, 2006.
Novel fluorogenic DNA probes are described. The probes (called Pleiades) have a minor groove binder (MGB) and a fluorophore at the 5'-end and a non-fluorescent quencher at the 3'-end of the DNA sequence. This configuration provides surprisingly low background and high hybridization-triggered fluorescence. Here, we comparatively study the performance of such probes, MGB-Eclipse probes, and molecular beacons. Unlike the other two probe formats, the Pleiades probes have low, temperature-independent background fluorescence and excellent signal-to-background ratios. The probes possess good mismatch discrimination ability and high rates of hybridization. Based on the analysis of fluorescence and absorption spectra we propose a mechanism of action for the Pleiades probes. First, hydrophobic interactions between the quencher and the MGB bring the ends of the probe and, therefore, the fluorophore and the quencher in close proximity. Second, the MGB interacts with the fluorophore and independent of the quencher is able to provide a modest (2–4-fold) quenching effect. Joint action of the MGB and the quencher is the basis for the unique quenching mechanism. The fluorescence is efficiently restored upon binding of the probe to target sequence due to a disruption in the MGB–quencher interaction and concealment of the MGB moiety inside the minor groove.
Present address: Sergey G. Lokhov, Cepheid, 1631 220th Street SE, Suite 101, Bothell, WA 98021, USA; Mikhail A. Podyminogin, Integrated DNA Technologies, Inc., 1710 Commercial Park, Coralville, IA 52241, USA
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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