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Nucleic Acids Research Advance Access published online on September 26, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm671
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© 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.

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Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature

Haukur Gudnason1, Martin Dufva1, D.D. Bang2 and Anders Wolff1,*

1Department of Micro- and Nanotechnology, Technical University of Denmark, bldg. 345, DK-2800 Lyngby and 2Laboratory of Applied Micro-nanotechnology, Department of Poultry, Fish, and Fur Animals, The National Veterinary Institute, Technical University of Denmark, Hangovej 2, DK-8200 Aarhus N, Denmark

*To whom correspondence should be addressed. Tel:+45 45256305; Fax: +45 45887762; Email: aw{at}mic.dtu.dk

Received April 29, 2007. Revised August 15, 2007. Accepted August 16, 2007.

The importance of real-time polymerase chain reaction (PCR) has increased steadily in clinical applications over the last decade. Many applications utilize SYBR Green I dye to follow the accumulation of amplicons in real time. SYBR Green I has, however, a number of limitations that include the inhibition of PCR, preferential binding to GC-rich sequences and effects on melting curve analysis. Although a few alternative dyes without some of these limitations have been recently proposed, no large-scale investigation into the properties of intercalating dyes has been performed. In this study, we investigate 15 different intercalating DNA dyes for their inhibitory effects on PCR, effects on DNA melting temperature and possible preferential binding to GC-rich sequences. Our results demonstrated that in contrast to the results of SYBR Green I, two intercalating dyes SYTO-13 and SYTO-82 do not inhibit PCR, show no preferential binding to GC rich sequences and do not influence melting temperature, Tm, even at high concentrations. In addition, SYTO-82 demonstrated a 50-fold lower detection limit in a dilution series assay. In conclusion, the properties of SYTO-82 and SYTO-13 will simplify the development of multiplex assays and increase the sensitivity of real-time PCR.


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