Capillary electrophoresis of double‐stranded DNA fragments using a new fluorescence intercalating dye EvaGreen

Abstract

EvaGreen is a new DNA intercalating dye successfully used in quantitative real-time PCR. In the present work, we firstly apply EvaGreen to the analysis of dsDNA by CE with LIF detection. Comparisons of EvaGreen dye with the commonly used dyes SYBR Green I and SYBR Gold were preformed in dsDNA analysis by CE. The linear range of dsDNA using EvaGreen was slightly wider than that using SYBR Gold and SYBR Green I, and the detection limits of dsDNA were not significantly different for the three dyes. Good separations of dsDNA fragments were obtained using the three dyes. Reproducibility of migration time and the peak area of dsDNA fragments with EvaGreen were better than those for SYBR Green I and SYBR Gold. The RSD values were 0.24-0.27% for migration time and 3.45-7.59% for peak area within the same day, 1.35-1.63% for migration time and 6.72-12.05% for peak area for three days. Our data demonstrated that EvaGreen is well suited for the dsDNA analysis by CE with LIF detection.

Facilitating aptamer selection and collection by capillary transient isotachophoresis with laser-induced fluorescence detection

“…However, differences in migration time for the labeled aptamer in on-column vs. pre-column labeling experiments were not accompanied by any significant difference in the reproducibility of migration times for a set of replicate runs conducted under these different labeling conditions (RSD = 1.3% and 0.3%, n = 4, for on-column and pre-column methods, respectively). Work conducted by others [32] established that the reproducibility of SYBR Gold labeling of dsDNA is largely independent of the number of DNA bases (from 72-234 bp). Thus, it is expected that the good reproducibility in peak area for SYBR Gold labeled ssDNA samples in this work could be extended to ssDNA samples of other lengths.…”

“…SYBR Gold is an asymmetrical cyanine dye that is typically used as a nucleic acid gel stain, but has been shown to successfully and efficiently bind ssDNA with greater sensitivity ( F = 0.7) than observed for other comparable dyes [27][28][29][30]. Previous studies have shown the labeling of doublestranded DNA with SYBR Gold for analysis by CE-LIF [31,32], but to the best of our knowledge, no studies have been conducted for the SYBR Gold labeling of ssDNA in CE-LIF. ctITP is an on-column, preconcentration and focusing technique that has been shown to improve concentration detection limits and increase the amount of sample that can be injected onto the capillary without loss of resolution of sample components [33][34][35].…”

A Cost‐effective High‐resolution Melting Approach using the EvaGreen Dye for DNA Polymorphism Detection and Genotyping in Plants

“…However, the high cost of these dyes often limits the application of this technology, in particular when dealing with a large number of samples. EvaGreen (Biotium Inc., Hayward, CA, USA) is a newly developed and low‐cost dye, and has been used in quantitative real‐time polymerase chain reaction (PCR), post‐PCR DNA melt curve analysis and several other applications (Ihrig et al 2006; Sang and Ren 2006; Wang et al 2006; White et al 2007; Akiyama et al 2009; Dagar et al 2009; Sun et al 2010; Ujino‐Ihara et al 2010). It is also a so‐called saturated dye that intercalates every single nucleotide of the double‐stranded DNA.…”

Development of Molecular Markers for the Determination of the New Cultivar 'Chunpoong' in Panax ginseng C. A. MEYER Associated with a Major Latex-Like Protein Gene

“…Moreover, we used EvaGreen dye to do the melting curve assay because EvaGreen has wider linear ranges in quantitative analysis and better reproducibility in the separations of dsDNA fragments compared with SYBR Green I. 22) In ginseng molecular marker research, this is the first report on the application of an allele-specific marker in real-time PCR for assaying numerous samples.…”