title: 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.
authors: Laboratory of Foodomics, CIAL (CSIC), Madrid, Spain.
authors: Alberto
authors: A
authors: Valdés
authors: Virginia
authors: V
authors: García-Cañas
authors: Alejandro
authors: A
authors: Cifuentes
mesh_terms: D001583:Benzoxazoles / Q000032:analysis*; D045744:Cell Line, Tumor; D004247:DNA / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000191:economics / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D007834:Lasers; D057230:Limit of Detection; D016133:Polymerase Chain Reaction; D011804:Quinolines / Q000032:analysis*; D011806:Quinolinium Compounds; D015203:Reproducibility of Results; D013997:Time Factors
chemical_list: D001583:Benzoxazoles; D005456:Fluorescent Dyes; D011804:Quinolines; D011806:Quinolinium Compounds; C089813:YO-PRO 1; D004247:DNA