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

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...

Full description

Bibliographic Details
Authors: Valdés, Alberto, García-Cañas, Virginia, Cifuentes, Alejandro
Format: article
Status:Versión aceptada para publicación
Publication Date:2013
Country:España
Institution:Consejo Superior de Investigaciones Científicas (CSIC)
Repository:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/99861
Online Access:http://hdl.handle.net/10261/99861
Access Level:Open access
Keyword:DNA separation
Fluorescent DNA-staining dye
GelGreen
PCR
CGE-LIF
id ES_c2f04cedbc763e9ae26d48c802ac537b
oai_identifier_str oai:digital.csic.es:10261/99861
network_acronym_str ES
network_name_str España
repository_id_str
spelling CGE-laser induced fluorescence of double-stranded DNA fragments using GelGreen dyeValdés, AlbertoGarcía-Cañas, VirginiaCifuentes, AlejandroDNA separationFluorescent DNA-staining dyeGelGreenPCRCGE-LIFNowadays, 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.Funded by: Ministerio de Economía y Competitividad, Spain. Grant Number: AGL2011-29857-C03-01 and Ministerio de Educación y Ciencia, Spain. Grant Number: CSD2007-00063.Peer reviewedPeer ReviewedWiley-VCHMinisterio de Economía y Competitividad (España)Ministerio de Educación y Ciencia (España)Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2014201420132014info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Postprintinfo:eu-repo/semantics/acceptedVersionapplication/pdfhttp://hdl.handle.net/10261/99861reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)InglésInglésThe underlying dataset has been published as supplementary material of the article in the publisher platform at DOI 10.1002/elps.201200624https://doi.org/10.1002/elps.201200624Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/998612026-05-22T06:33:51Z
dc.title.none.fl_str_mv CGE-laser induced fluorescence of double-stranded DNA fragments using GelGreen dye
title CGE-laser induced fluorescence of double-stranded DNA fragments using GelGreen dye
spellingShingle CGE-laser induced fluorescence of double-stranded DNA fragments using GelGreen dye
Valdés, Alberto
DNA separation
Fluorescent DNA-staining dye
GelGreen
PCR
CGE-LIF
title_short CGE-laser induced fluorescence of double-stranded DNA fragments using GelGreen dye
title_full CGE-laser induced fluorescence of double-stranded DNA fragments using GelGreen dye
title_fullStr CGE-laser induced fluorescence of double-stranded DNA fragments using GelGreen dye
title_full_unstemmed CGE-laser induced fluorescence of double-stranded DNA fragments using GelGreen dye
title_sort CGE-laser induced fluorescence of double-stranded DNA fragments using GelGreen dye
dc.creator.none.fl_str_mv Valdés, Alberto
García-Cañas, Virginia
Cifuentes, Alejandro
author Valdés, Alberto
author_facet Valdés, Alberto
García-Cañas, Virginia
Cifuentes, Alejandro
author_role author
author2 García-Cañas, Virginia
Cifuentes, Alejandro
author2_role author
author
dc.contributor.none.fl_str_mv Ministerio de Economía y Competitividad (España)
Ministerio de Educación y Ciencia (España)
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv DNA separation
Fluorescent DNA-staining dye
GelGreen
PCR
CGE-LIF
topic DNA separation
Fluorescent DNA-staining dye
GelGreen
PCR
CGE-LIF
description 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.
publishDate 2013
dc.date.none.fl_str_mv 2013
2014
2014
2014
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Postprint
info:eu-repo/semantics/acceptedVersion
format article
status_str acceptedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/99861
url http://hdl.handle.net/10261/99861
dc.language.none.fl_str_mv Inglés
Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv The underlying dataset has been published as supplementary material of the article in the publisher platform at DOI 10.1002/elps.201200624
https://doi.org/10.1002/elps.201200624
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Wiley-VCH
publisher.none.fl_str_mv Wiley-VCH
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869418730919821312
score 15.812429

Similar Items