Figure 2. A comparison of the raw fluorescence signal from qPCR reactions performed with two EvaGreen®-based master mixes (Forget-Me-Not™ EvaGreen® and Fast EvaGreen®) and QuantiNova® SYBR® Green I. EvaGreen® Dye is less inhibitory than SYBR® Green I, allowing for a much brighter signal.
Biotium
Unveiling the Full Potential of LAMP
Loop-mediated isothermal amplification’s (LAMP) simplicity and speed make the method particularly attractive for point-of-care diagnostics in resource-limited contexts. It also shines in field settings where it’s used for rapid detection of plant pathogens or infectious disease agents like malaria, tuberculosis, and SARS-CoV-2.10, 11 Both LAMP and PCR amplify DNA, but there are a few key differences:
Temperature: The strand-displacing properties of Bst DNA polymerase used in LAMP allows for rapid amplification of a specific sequence at one temperature (60-65° C). Standard PCR requires cycling between multiple temperatures to denature the DNA, anneal primers, and extend the new strand.
Generated product: PCR generates many copies of discrete, single-sized amplicons while LAMP creates long concatemers of the same repeated sequence.
Reaction duration: PCR thermocycling protocols often take at least 1.5 hours, while LAMP reactions are complete in 30-40 minutes.
Because of SYBR® Green I’s inhibitory effect on DNA polymerization at higher concentrations, it’s mainly used as an end point reporter in LAMP.4 As summarized by Fishbach et al. , EvaGreen® is more suited for real-time monitoring of LAMP reactions as it does not impact DNA polymerization and, therefore, can be present throughout the entirety of the reaction.4, 7, 8
Increasing Precision in High-Resolution Melting (HRM®) Experiments
HRM® is a post-PCR method for analyzing the melt curves of amplified DNA fragments. The method requires a DNA binding dye like EvaGreen®, and an instrument with exceptional thermal sensitivity that can distinguish melt curves between fragments with small sequence variations. The analysis can be used for DNA mapping, mutation scanning, species identification, zygosity testing, DNA fingerprinting, and more.
Despite its popularity, SYBR® Green I has been found to be unreliable for HRM® due to problems caused by dye redistribution at higher concentrations. EvaGreen® was designed to be less inhibitory in the PCR reactions, permitting the use of saturating dye concentration for maximal signal and better high-resolution DNA melt analysis with less dye redistribution. 5,6
The only DNA dye suitable for multiplex ddPCR
Droplet digital PCR (ddPCR) is a digital PCR method that is based on water-oil emulsion droplet technology. The sample is fractionated into tens of thousands of droplets, which act as individual test tubes or wells in a plate in which the PCR reaction takes place. The technique allows researchers to multiplex targets from the same sample and requires smaller sample amounts than other commercially available digital PCR systems. This results in lower sample and reagent volume requirements and reduced overall cost while maintaining the sensitivity and precision of digital PCR. Digital PCR techniques, like ddPCR, also offer the ability for absolute quantification of molecules.
TaqMan® probes have traditionally been used for this technique, but their expense and the required complexity in assay design are significant drawbacks. EvaGreen® is less likely to inhibit PCR and promote mispriming than competitor DNA-binding dyes, and it shows superior performance when used in qPCR and for high resolution melt curve analysis. This makes it a promising tool for ddPCR. McDermott et al. confirmed its effectiveness in their 2013 paper in which they identified EvaGreen® as the only DNA-binding dye validated for ddPCR in their experimental system. In this study, they also found that EvaGreen® increased resolution and allowed for the quantification of multiple target species in a single well of droplets.9
A Breakthrough in Safety
DNA-binding dyes are inherently dangerous due to their potential to cause mutations. Ohta et al. found SYBR® Green I to be even more environmentally toxic than ethidium bromide, a well-known mutagen.13 No safety data currently exists on other common PCR and HRM dyes (e.g., SYTO®9, LCGreen®, BRYT Green®, and ResoLight). However, these dyes are known for quickly permeating cells and posing a potential risk of genotoxicity. This motivated Biotium scientists to create EvaGreen®, a dye that is impenetrable to latex gloves and cell membranes, with metabolites that have little or no interaction with DNA. Testing by Biotium and independent testing services confirmed that it is noncytotoxic and nonmutagenic at concentrations well above the working concentrations used in qPCR.
References
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SYBR and SYTO are registered trademarks of Thermo Fisher Scientific; LCGreen is a registered trademark of Biofire; BRYT Green is a registered trademark of Promega; HRM is a registered trademark of Idaho Technologies; QuantiNova is a registered trademark of Qiagen.
This story was originally published by Biotium .