title: Comparison of different DNA binding fluorescent dyes for applications of high-resolution melting analysis.
abstract: OBJECTIVE
  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.
  
  
  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: 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.
authors: Jan
authors: J
authors: Radvanszky
authors: Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Mlynska dolina, 842 15 Bratislava 4, Slovakia.
authors: Milan
authors: M
authors: Surovy
authors: Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Mlynska dolina, 842 15 Bratislava 4, Slovakia.
authors: Emilia
authors: E
authors: Nagyova
authors: 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.
authors: Gabriel
authors: G
authors: Minarik
authors: 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.
authors: Ludevit
authors: L
authors: Kadasi
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry*; D060005:Genotyping Techniques; D009691:Nucleic Acid Denaturation; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D005456:Fluorescent Dyes; D004247:DNA
keywords: Fluorescent dyes; Genotyping; HRM; High-resolution melting; Screening