CGE‐laser induced fluorescence of double‐stranded DNA fragments using GelGreen dye

Abstract

Nowadays, new solutions focused on the replacement of reagents hazardous to human health are highly demanded in laboratories and Green Chemistry. In the present work, GelGreen, a new nonhazardous DNA staining reagent, has been assayed for the first time to analyze double‐stranded DNA by CGE with LIF detection. The effect of GelGreen concentration on S/N ratio and migration time of a wide concentration range of standard DNA mixtures was evaluated. Under optimum GelGreen concentration in the sieving buffer efficient and sensitive separations of DNA fragments with sizes from 100–500 base pairs (bp) were obtained. A comparison in terms of resolution, time of analysis, LOD, LOQ, reproducibility, sizing performance, and cost of analysis was established between two optimized CGE‐LIF protocols for DNA analysis, one based on the dye YOPRO‐1 (typically used for CGE‐LIF of DNA fragments) and another one using the new GelGreen. Analyses using YOPRO‐1 were faster than those using GelGreen (ca. 31 min versus 34 min for the analysis of 100–500 bp DNA fragments). On the other side, sensitivity using GelGreen was twofold higher than that using YOPRO‐1. The cost of analysis was significantly cheaper (ninefold) using GelGreen than with YOPRO‐1. The resolution values and sizing performance were not significantly different between the two dyes (e.g. both dyes allowed the separation of fragments differing in only 2 bp in the 100–200 bp range). The usefulness of the separation method using GelGreen is demonstrated by the characterization of different amplicons obtained by PCR.

Recent advances in the application of capillary electromigration methods for food analysis and Foodomics

“…Last developments in CGE include novel sieving matrices , novel capillary coatings , and additives to provide high resolution and miniaturization into microchips . A recent example of these innovative solutions in DNA separations is represented by the employment of the nonhazardous DNA staning reagent GelGreen in CGE‐LIF . Using this dye, detection of DNA fragments exhibited twofold improved sensitivity at lower costs in comparison with the conventionally used YOPRO‐1 dye.…”

Capillary Electrophoresis Based on Nucleic Acid Detection as Used in Food Analysis

“…Consequently, fluorescence detection has been used in DNA analysis to overcome the low sensitivity of CE (Tseng and others ; Ban and Song ; de Kort and others ; Ruan and others ). However, this solution demands labeling samples with a fluorescent marker or intercalating dyes (Hernández‐Chávez and others ; Valdes and others ). Unfortunately, these procedures are time‐consuming and expensive, and most dyes are toxic (such as ethidium bromide).…”

Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease

“…Consequently, fluorescence detection becomes the solution for the sensitivity challenges of DNA analysis used by most researchers [26,135,141]. However, this solution demands labeling samples with a fluorescence marker or intercalating dyes [142]. Unfortunately, these procedures are time consuming and expensive, and most dyes are toxic (such as ethidium bromide).…”