title: Quantitative, small-scale, fluorophore-assisted carbohydrate electrophoresis implemented on a capillary electrophoresis-based DNA sequence analyzer.
abstract: Fluorophore-assisted carbohydrate electrophoresis (FACE) is an analytical method for characterizing carbohydrate chain length that has been applied to neutral, charged, and N-linked oligosaccharides and that has been implemented using diverse separation platforms, including polyacrylamide gel electrophoresis and capillary electrophoresis. In this article, we describe three substantial improvements to FACE: (i) reducing the amount of starch and APTS required in labeling reactions and systematically analyzing the effect of altering the starch and 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) concentrations on the reproducibility of the FACE peak area distributions; (ii) implementing FACE on a multiple capillary DNA sequencer (an ABI 3130xl), enabling higher throughput than is possible on other separation platforms; and (iii) developing a protocol for producing quantitative output of peak heights and areas using genetic marker analysis software. The results of a designed experiment to determine the effect of decreasing both the starch and fluorophore concentrations on the sensitivity and reproducibility of FACE electrophoregrams are presented. Analysis of the peak area distributions of the FACE electrophoregrams identified the labeling reaction conditions that resulted in the smallest variances in the peak area distributions while retaining strong fluorescence signals from the capillary-based DNA sequencer.
authors.affiliation: New Zealand Institute for Plant & Food Research, Christchurch 8140, New Zealand.
authors.forename: Sarah
authors.initials: S
authors.lastname: Murray
authors.forename: Marian
authors.initials: M
authors.lastname: McKenzie
authors.forename: Ruth
authors.initials: R
authors.lastname: Butler
authors.forename: Samantha
authors.initials: S
authors.lastname: Baldwin
authors.forename: Kevin
authors.initials: K
authors.lastname: Sutton
authors.forename: Ian
authors.initials: I
authors.lastname: Batey
authors.forename: Gail M
authors.initials: GM
authors.lastname: Timmerman-Vaughan
mesh_terms: D000818:Animals; D002236:Carbohydrate Conformation; D016009:Chi-Square Distribution; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000295:instrumentation; D013194:Staining and Labeling; D013213:Starch / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D013213:Starch