Jan Radvanszky1, Milan Surovy2, Emilia Nagyova2, Gabriel Minarik3, Ludevit Kadasi4. 1. Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlarska 5, 833 34 Bratislava, Slovakia; Geneton s.r.o., Cabanova 14, 841 02 Bratislava, Slovakia; Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Mlynska dolina, 842 15 Bratislava 4, Slovakia. Electronic address: jradvanszky@gmail.com. 2. Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Mlynska dolina, 842 15 Bratislava 4, Slovakia. 3. Geneton s.r.o., Cabanova 14, 841 02 Bratislava, Slovakia; Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Mlynska dolina, 842 15 Bratislava 4, Slovakia; Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Sasinkova 4, 811 08 Bratislava, Slovakia. 4. Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlarska 5, 833 34 Bratislava, Slovakia; Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Mlynska dolina, 842 15 Bratislava 4, Slovakia.
Abstract
OBJECTIVES: Different applications of high-resolution melting (HRM) analysis have been adopted for a wide range of research and clinical applications. This study compares the performance of selected DNA binding fluorescent dyes for their possible application in HRM. DESIGN AND METHODS: We compared twelve dyes with basic properties considered relevant for PCR amplification and melting curve analysis. These included PCR inhibition, fluorescence intensity, the ability to generate melting curves and their effect on melting temperature (Tm). Seven of these dyes with promising properties were then evaluated for possible use in basic HRM applications; such as small amplicon genotyping, genotyping of a 1 kb insertion/deletion polymorphism, probe-based genotyping and mutation screening. RESULTS: Five dyes failed to exhibit promising properties during the first part of the study, and these were excluded from further testing. Of the remaining dyes, SYTO11, SYTO13 and SYTO16 showed better PCR inhibitory and Tm affecting properties compared to commercial HRM dyes LCGreen Plus, EvaGreen and ResoLight. Although the SYTO dyes generally exhibited good discrimination powers in HRM applications, SYTO11 and SYTO14 gave low signal intensity and lower quality results. CONCLUSIONS: Our results suggest that the best performing dyes for HRM are those commercially offered for HRM analyses. However, the performance of SYTO16 and SYTO13 was comparable to the HRM dyes in the majority of our assays, thus demonstrating that they are also quite suitable for both real-time PCR and HRM applications.
OBJECTIVES: Different applications of high-resolution melting (HRM) analysis have been adopted for a wide range of research and clinical applications. This study compares the performance of selected DNA binding fluorescent dyes for their possible application in HRM. DESIGN AND METHODS: We compared twelve dyes with basic properties considered relevant for PCR amplification and melting curve analysis. These included PCR inhibition, fluorescence intensity, the ability to generate melting curves and their effect on melting temperature (Tm). Seven of these dyes with promising properties were then evaluated for possible use in basic HRM applications; such as small amplicon genotyping, genotyping of a 1 kb insertion/deletion polymorphism, probe-based genotyping and mutation screening. RESULTS: Five dyes failed to exhibit promising properties during the first part of the study, and these were excluded from further testing. Of the remaining dyes, SYTO11, SYTO13 and SYTO16 showed better PCR inhibitory and Tm affecting properties compared to commercial HRM dyes LCGreen Plus, EvaGreen and ResoLight. Although the SYTO dyes generally exhibited good discrimination powers in HRM applications, SYTO11 and SYTO14 gave low signal intensity and lower quality results. CONCLUSIONS: Our results suggest that the best performing dyes for HRM are those commercially offered for HRM analyses. However, the performance of SYTO16 and SYTO13 was comparable to the HRM dyes in the majority of our assays, thus demonstrating that they are also quite suitable for both real-time PCR and HRM applications.
Authors: Zuzana Pös; Ondrej Pös; Jakub Styk; Angelika Mocova; Lucia Strieskova; Jaroslav Budis; Ludevit Kadasi; Jan Radvanszky; Tomas Szemes Journal: Int J Mol Sci Date: 2020-11-16 Impact factor: 5.923