Comparison of different DNA binding fluorescent dyes for applications of high-resolution melting analysis

Radvánszky, Ján; Surovy, Milan; Nagyová, Emília; Minárik, Gabriel; Kádaši, Ľudevít

doi:10.1016/j.clinbiochem.2015.01.010

Cited by 29 publications

(14 citation statements)

References 34 publications

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“…This is in contrast to DD, which showed inhibition at concentrations >1.5. In the same study, EG showed no inhibition of qPCR at concentrations >2, with DNA amounts ranging 25100 ng (12), indicating EG has less of an inhibitory effect on qPCR when compared with DD.…”

Section: Resultsmentioning

confidence: 89%

“…A previous study (12) showed that at lower DNA quantities (25 ng), SG can inhibit the reaction when concentrations are >0.5. This is in contrast to DD, which showed inhibition at concentrations >1.5.…”

Section: Resultsmentioning

confidence: 97%

“…SG is known to inhibit PCR at high concentrations and, with 25 ng of DNA, was shown to inhibit the reaction at concentrations >1. In comparison, EG still shows amplification at a concentration of 2.5, indicating that EG inhibits PCR less than SG (12). …”

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confidence: 99%

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Optimization of Diamond Nucleic Acid Dye for Quantitative PCR

2016

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Here, we evaluate Diamond Nucleic Acid Dye (DD) for use in quantitative PCR (qPCR) applications. Although DD is a commercially available stain for detection of DNA separated by gel electrophoresis, its use as a detection dye in qPCR has yet to be described. To determine if DD can be used in qPCR, we investigated its inhibitory effects on qPCR at concentrations ranging 0.1-2.5×. Serial dilution of DNA was used to determine the efficiency, sensitivity, and linearity of DD-generated qPCR data in comparison to other commonly used fluorescent dyes such as SYBR Green (SG), EvaGreen (EG), and BRYT Green (BG). DD was found to be comparable with other dyes for qPCR applications, with an R2 value >0.9 and an efficiency of 0.83. Mitochondrial DNA (mtDNA) target signals were successfully produced by DD over a DNA dilution range of ~28 ng- 0.28 pg, demonstrating comparable sensitivity to the other dyes investigated. Cq values obtained using DD were lower than those using EG by almost 7 cycles. We conclude that Diamond Nucleic Acid Dye is a cheaper, less toxic alternative for qPCR applications.

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Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 97%

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Optimization of Diamond Nucleic Acid Dye for Quantitative PCR

2016

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“…High levels of DNA saturation is important for melt curve applications, especially high resolution melting analysis (HRM), where small differences in the amplicon melt pattern are used to infer genotypes [15]. Interestingly, RL and EG, the two dyes that showed the greatest losses in FI and substantial changes of Tm in presence of HA, are commonly used and recommended for HRM [16], [17]. The blend of SY/SG and SY alone on the other hand, gave the smallest differences in Tm when HA was added.…”

Section: Resultsmentioning

confidence: 99%

Blending DNA binding dyes to improve detection in real-time PCR

Jansson

Koliana

Sidstedt

et al. 2017

Biotechnology Reports

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HighlightsHumic acid quenches the fluorescence of the dsDNA binding dyes EvaGreen, ResoLight, SYBR Green I and SYTO9.A four-dye blend containing the dyes above provides maximum fluorescence signals in presence and absence of humic acid.Blending complementary dyes enables improved qualitative detection of DNA in samples with high amounts of humic acid.Humic acid interactions with dsDNA binding dyes lower the Tm of amplicons, thereby disturbing melt curve analysis.

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“…HRM was selected since it represents a method that is increasingly popular and widespread due to its accuracy, easy, fast, and cost effective application. It has recently been also successfully adopted in a series of research applications that include single base change genotyping, insertion and deletion genotyping sequence matching, methylation profiling, internal tandem duplication detection, and mutation screening (Radvanszky et al, 2015), while it is in its infancy for the detection of mutations associated with the resistance of fungal plant pathogens to fungicides.…”

Section: Discussionmentioning

confidence: 99%

Detection of sdhB Gene Mutations in SDHI-Resistant Isolates of Botrytis cinerea Using High Resolution Melting (HRM) Analysis

2016

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Botrytis cinerea, is a high risk pathogen for fungicide resistance development. Pathogen’ resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I, and H272L/R/Y mutations. Based on the sequence of sdhB subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant’s DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the four mutations located at codon 225, the N230I mutation, the three mutations located in codon 272, and the non-mutated isolates (isolates of wild-type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdhB mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping-off symptoms. HRM analysis data were compared with a standard PIRA–PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I, and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate, and rapid identification of several sdhB mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of anti-resistance strategies implemented in crops heavily treated with botryticides.

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Comparison of different DNA binding fluorescent dyes for applications of high-resolution melting analysis

Cited by 29 publications

References 34 publications

Optimization of Diamond Nucleic Acid Dye for Quantitative PCR

Optimization of Diamond Nucleic Acid Dye for Quantitative PCR

Blending DNA binding dyes to improve detection in real-time PCR

Detection of sdhB Gene Mutations in SDHI-Resistant Isolates of Botrytis cinerea Using High Resolution Melting (HRM) Analysis

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