Journal article
Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature
BioLabChip Group, LabChip Section, Department of Micro- and Nanotechnology, Technical University of Denmark1
LabChip Section, Department of Micro- and Nanotechnology, Technical University of Denmark2
Department of Micro- and Nanotechnology, Technical University of Denmark3
Micro Array Technology, Department of Micro- and Nanotechnology, Technical University of Denmark4
Section of Poultry Diseases, Division of Poultry, Fish and Fur Animals, National Veterinary Institute, Technical University of Denmark5
Division of Poultry, Fish and Fur Animals, National Veterinary Institute, Technical University of Denmark6
National Veterinary Institute, Technical University of Denmark7
Cell Particle Handling, Department of Micro- and Nanotechnology, Technical University of Denmark8
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, T-m, 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.
Language: | English |
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Publisher: | Oxford University Press |
Year: | 2007 |
Pages: | e127 |
ISSN: | 13624962 and 03051048 |
Types: | Journal article |
DOI: | 10.1093/nar/gkm671 |
ORCIDs: | Dufva, Hans Martin , Bang, Dang Duong and Wolff, Anders |