Improved stability and electrophoretic properties of preformed fluorescent cationic dye-DNA complexes in a taps-tetrapentylammonium buffer in agarose slab gels

Anal Biochem. 1997 Oct 1;252(1):110-4. doi: 10.1006/abio.1997.2303.

Abstract

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-NPe+4) 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 NH+4. We report here the behavior of preformed double-stranded DNA-dye complexes on agarose slab gel electrophoresis in 40 mM Taps-NPe+4, 1 mM H2EDTA, 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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Buffers
  • DNA / chemistry*
  • DNA / metabolism
  • Electrophoresis, Agar Gel / methods*
  • Fluorescent Dyes / chemistry*
  • Fluorescent Dyes / metabolism
  • Quaternary Ammonium Compounds / chemistry
  • Sulfonic Acids / chemistry

Substances

  • Buffers
  • Fluorescent Dyes
  • Quaternary Ammonium Compounds
  • Sulfonic Acids
  • 3-(tris(hydroxymethyl)methylamino)-1-propanesulfonic acid
  • DNA
  • tetrapentylammonium