Gudnason Haukur, Dufva Martin, Bang D D, Wolff Anders
Department of Micro- and Nanotechnology, Technical University of Denmark, bldg. 345, DK-2800 Lyngby, Denmark.
Nucleic Acids Res. 2007;35(19):e127. doi: 10.1093/nar/gkm671. Epub 2007 Sep 26.
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.
在过去十年中,实时聚合酶链反应(PCR)在临床应用中的重要性稳步提升。许多应用利用SYBR Green I染料实时追踪扩增子的积累。然而,SYBR Green I存在一些局限性,包括抑制PCR、优先结合富含GC的序列以及对熔解曲线分析的影响。尽管最近有人提出了一些没有这些局限性的替代染料,但尚未对嵌入染料的特性进行大规模研究。在本研究中,我们研究了15种不同的嵌入DNA染料对PCR的抑制作用、对DNA熔解温度的影响以及与富含GC序列的可能优先结合情况。我们的结果表明,与SYBR Green I的结果不同,两种嵌入染料SYTO-13和SYTO-82不抑制PCR,不优先结合富含GC的序列,即使在高浓度下也不影响熔解温度(Tm)。此外,在稀释系列测定中,SYTO-82的检测限低50倍。总之,SYTO-82和SYTO-13的特性将简化多重检测的开发并提高实时PCR的灵敏度。