Improved Stability and Electrophoretic Properties of Preformed Fluorescent Cationic Dye–DNA Complexes in a Taps–Tetrapentylammonium Buffer in Agarose Slab Gels
✍ Scribed by Zhaoxian Zeng; Steven M. Clark; Richard A. Mathies; Alexander N. Glazer
- Publisher
- Elsevier Science
- Year
- 1997
- Tongue
- English
- Weight
- 123 KB
- Volume
- 252
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Glazer and co-workers (1-5) have shown that various homo-and heterodimeric bisintercalators, with the High-resolution capillary electrophoresis sizing of preformed complexes of bis-intercalating fluores-intercalating moieties bridged by a polyalkylamino cent dyes with double-stranded DNA has been dem-linker, form noncovalent complexes with doubleonstrated using hydroxyethylcellulose and 3-[trisstranded DNA (dsDNA) 5 which are stable under the (hydroxymethyl)methylamino]-1-propanesulfonic usual conditions of slab gel electrophoresis. Such preacid -tetrapentylammonium (Taps -NPe / 4 ) buffers formed dsDNA-dye complexes have been successfully (S. M. Clark and R. A. Mathies, Anal. Chem. 69, 1355exploited in electrophoretic applications to size dsDNA 1363, 1997). Such capillary electrophoresis separarestriction fragments (2, 3, 5, 6) and to study specific tions were unattainable in conventional buffers conprotein-dsDNA complexes (7, 8). In the course of these taining other cations such as Tris / , Na / , and NH / 4 . studies, it was noted that the mobility of dsDNA-dye We report here the behavior of preformed doublecomplexes is dependent on the dye: DNA base pairs (bp) stranded DNA -dye complexes on agarose slab gel ratio and that at dye:bp ratios ú1:50, some transfer of electrophoresis in 40 mM Taps -NPe / 4 , 1 mM H 2 EDTA, dye between labeled and unlabeled dsDNA molecules pH 8.2. Upon electrophoresis in this buffer (a) comtakes place (1, 6). These problems were successfully plexes formed at DNA base pairs:dye ratios ranging addressed where the DNA concentration of the samples from 100:1 to 5:1 show the same mobility; (b) the halfunder study was known in advance and where comlives of DNA -dye complexes with monointercalators plexes were formed at the appropriate dye-dsDNA bp are two-to threefold longer than those in commonly ratio (4-6). However, these considerations make it used Tris buffers; (c) there is little dye transfer besomewhat more difficult to use preformed DNA-dye