Improved Stability and Electrophoretic Properties of Preformed Fluorescent Cationic Dye–DNA Complexes in a Taps–Tetrapentylammonium Buffer in Agarose Slab Gels

• Published in: Analytical biochemistry 1997-10, Vol.252 (1), p.110-114
• Main Authors: Zeng, Zhaoxian, Clark, Steven M., Mathies, Richard A., Glazer, Alexander N.
• Format: Article
• Language: English
• Subjects: Buffers; DNA - chemistry; DNA - metabolism; Electrophoresis, Agar Gel - methods; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; Quaternary Ammonium Compounds - chemistry; Sulfonic Acids - chemistry
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1997.2303

High-resolution capillary electrophoresis sizing of preformed complexes of bis-intercalating fluorescent dyes with double-stranded DNA has been demonstrated using hydroxyethylcellulose and 3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid–tetrapentylammonium (Taps–NPe4+) buffers (S. M. Clark and R. A. Mathies,Anal. Chem.69, 1355–1363, 1997). Such capillary electrophoresis separations were unattainable in conventional buffers containing other cations such as Tris+, Na+, and NH4+. We report here the behavior of preformed double-stranded DNA–dye complexes on agarose slab gel electrophoresis in 40 mmTaps–NPe4+, 1 mmH2EDTA, pH 8.2. Upon electrophoresis in this buffer (a) complexes formed at DNA base pairs:dye ratios ranging from 100:1 to 5:1 show the same mobility; (b) the half-lives of DNA–dye complexes with monointercalators are two- to threefold longer than those in commonly used Tris buffers; (c) there is little dye transfer between labeled and unlabeled DNA molecules; and (d) precise two-color sizing of preformed restriction fragment–dye complexes with fluorescent bisintercalators is achieved.