Effects of gel material on fluorescence lifetime detection of dyes and dye-labeled DNA primers in capillary electrophoresis.
Investigations of fluorescence lifetimes of the dye 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoic acid (NBD-HA) and of DNA M13 primers labeled with NBD-HA, Cy3, rhodamine green and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3-propionic acid (BODIPY-FL) dyes in polyacrylamide gels of various degrees of crosslinking and different crosslinkers, and in a cellulose sieving buffer with different organic modifiers, are described. The dependence of fluorescence lifetime on gel matrix and on experimental conditions was studied in order to identify which factors may be important for optimization of multiplex fluorescence lifetime detection. Lifetimes were determined in both batch solution and on-the-fly, on-column in CE. Results show that lifetimes of the primer-attached dyes remain constant in gels of different composition. Additionally, multiexponential fluorescence decays are observed for primer-attached dyes in batch solutions of the cellulose sieving buffers but are reduced to monoexponential decays when measured on-the-fly, on-column in CE. Lifetime detectability can be improved by addition of an organic modifier to the gel matrix.
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Department of Chemistry, P.M. Gross Chemical Laboratory, Duke University, Durham, NC 27708-0346, USA.
McGown
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D000180:Acrylic Resins; D001896:Boron Compounds / Q000737:chemistry; D002021:Buffers; D002232:Carbocyanines / Q000737:chemistry; D002482:Cellulose; D017931:DNA Primers / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D005782:Gels; D013995:Time
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D000180:Acrylic Resins; D001896:Boron Compounds; D002021:Buffers; D002232:Carbocyanines; D017931:DNA Primers; D005456:Fluorescent Dyes; D005782:Gels; C089187:cyanine dye 3; C016680:polyacrylamide gels; D002482:Cellulose