Related Experiment Video

Updated: Feb 15, 2026

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Published on: November 5, 2010

Characterization of the binding interactions between EvaGreen dye and dsDNA

L C T Shoute¹, G R Loppnow

¹Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada. glen.loppnow@ualberta.ca.

Physical Chemistry Chemical Physics : PCCP

|January 31, 2018

Summary

EvaGreen dye exhibits positive cooperativity when binding to duplex DNA, with intercalation occurring at specific sites. At high concentrations, external binding and stacked chains form, disrupting intercalation.

Area of Science:

Biophysical Chemistry
Molecular Biology
Spectroscopy

Background:

EvaGreen (EG) dye is widely used in DNA analysis techniques like quantitative PCR and melting analysis.
Understanding the binding mechanism of EG with double-stranded DNA (dsDNA) is crucial for optimizing these applications.
Previous studies suggest complex interactions between dyes and DNA, necessitating detailed investigation.

Purpose of the Study:

To determine the binding isotherm of EvaGreen dye with duplex DNA (poly-dA17·poly-dT17).
To elucidate the binding mechanism, including cooperativity and site occupancy.
To investigate the effect of dye concentration on the binding mode.

Main Methods:

Spectroscopic analysis (absorption and fluorescence) of EvaGreen and its dsDNA complex.
Determination of binding isotherms.
Modeling of binding data using the Hill equation and Scatchard analysis with the modified McGhee and von Hippel model.

Main Results:

The binding isotherm is sigmoidal, indicating positive cooperative binding of EG to dsDNA.
Scatchard analysis confirms positive cooperativity, yielding an intrinsic binding constant of 3.6 × 10^5 M^-1.
A site occupancy of 4 base pairs and a cooperativity parameter of 8.1 were determined, suggesting intercalation with gaps.
At high EG concentrations, intercalation is disrupted, leading to external binding and formation of stacked chains.

Conclusions:

EvaGreen exhibits positive cooperative binding to duplex DNA, with intercalation occurring in a specific pattern.
The binding mode transitions at high dye concentrations, forming external stacked chains.
These findings provide critical insights into the molecular interactions of EvaGreen, relevant for its application in DNA analysis.

More Related Videos

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Published on: June 26, 2016

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

Published on: November 21, 2017

Related Concept Videos

The Equilibrium Binding Constant and Binding Strength

The Equilibrium Binding Constant and Binding Strength

The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:

The Equilibrium Binding Constant and Binding Strength

The Equilibrium Binding Constant and Binding Strength

Factors Affecting Protein-Drug Binding: Drug Interactions

Factors Affecting Protein-Drug Binding: Drug Interactions

Drug interactions are a critical aspect of pharmacology and can occur when two or more drugs compete for the same binding site. This competition can result in one drug displacing another, altering the effect of the displaced drug. Drug interactions are complex processes that rely heavily on how much of the displacer drug is present and how strongly it can bind to the same sites as the displaced drug.
Displacement interactions can have varying outcomes, ranging from toxicity to virtually...

Ligand Binding Sites

Ligand Binding Sites

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...

Conserved Binding Sites

Conserved Binding Sites

Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally...

Conserved Binding Sites

Conserved Binding Sites

Articles linked to this work by shared authors, journal, and citation graph.

UV Raman spectroscopy of hydrocarbons.

Philosophical transactions. Series A, Mathematical, physical, and engineering sciences·2004

Raman spectroscopy as a discovery tool in carbohydrate chemistry.

Analytical chemistry·2000

Identification of the quinone cofactor in mammalian semicarbazide-sensitive amine oxidase.

Biochemistry·1998

Structure of the retinal chromophore in 7,9-dicis-rhodopsin.

Biochemistry·1990

Why are blue visual pigments blue? A resonance Raman microprobe study.

Proceedings of the National Academy of Sciences of the United States of America·1989

Excited-state structure and isomerization dynamics of the retinal chromophore in rhodopsin from resonance Raman intensities.

Biophysical journal·1988

See all related articles