This thesis describes the development of methods for theseparation of DNA sequencing fragments and otheroligonucleotides by capillary gel electrophoresis (CGE), and isbased on live publications.
In the first work [Il, an eightfold increase in lifetime ofultra-narrow bore (2-lo-micron ID) gel filled columns comparedto 50-micron ID columns at high electrical field strengths wasexperienced, presumably due to the better heat dissipationobtained in the former columns. Subsequently, the lo-micron IDcolumns were employed for electrophoresis of fluoresceinisothiocyanate-labeled sequencing fragments.
The setond [II], and the third [III]publication deal withthe problem of intra-molecular hydrogen bonding duringseparation of DNA sequencing fragments. In publication numbertwo [II], a combined use of chemical denaturants and elevatedtemperature was effective in suppressing anomalies in fragmentmigration, and also, the total analysis time decreased.However, for cross linked gel columns the resolution betweenlong DNA fragments was reduced.
In publication number three [III], a cross-linkedpoly(dimethylacrylamide) sieving matrix prepared in an organicsolvent, N-methylformamide (NMF), was employed forelectrophoretic separations of oligonucleotides. Sieving anddenaturing properties similar to those obtained in aqueous,urea saturated, polyacrylamide gels were observed. Additionalresults of unpublished work with NMF based linear polymersolutions have been incorporated in the general part of thethesis.
In the fourth article [IV], a new technique is described forthe preparation of on-column detection windows inpolyimide-coated fused-silica capillary columns. A simpletreatment with fuming nitrit acid at room temperature isperformed, which does not damage columns filled within situpolymerized gels. The method can also beutilized for stripping the toating from an array of columns inone step.
In publication number tive [VI, the construction andevaluation of a fiber-optic cell for UV absorbance detection incapillary electrophoresis is described. Optical fibers with alarge core diameter were combined with focusing optics in orderto maximize light throughput and to reduce stray light. Thecompact and flexible design of the fiber-optic cell made itsuitable for high speed CGE, utilizing short columns, which isdemonstrated in the paper.
Keywords:capillary electrophoresis, capillary gelelectrophoresis, DNA separations, DNA sequencing,oligonucleotide analysis,DNA Sanger fragments, narrow-bore gelcolumns, cross linked polyacrylamide, polymerized gels,confocal laser-induced fluorescence detection, LIF detection,loop-structures in DNA, heat denaturation, poly(N-acryloylaminopropanol), poly(AAP), poly(dimethylacrylamide), poly(DMA),chemical denaturation, N-methylformamide, NMF, NMF based gels,non-aqueous gels, replaceable matrices, poly(ethyleneoxide)(PEO), hydroxethyl cellulose (HEC), poly(vinylpyrrolidone)(PVP), polyimide-toating removal, detection-windows,fiber-optics, absorbance detection cell, focusing optics,circular aperture.
© Peter Lindberg, 1998
Stockholm: Kemi , 1998. , 65 p.