SEPARATION OF DNA FRAGMENTS BY CAPILLARY ELECTROPHORESIS USING REPLACEABLE LINEAR POLYACRYLAMIDE MATRICES

The use of low percent (1.5-6%T) replaceable linear polyacrylamide (LP A) network matrices for rapid separation of double-stranded DNA fragme nts was explored. Separations of fragments ranging from 20 to 23 000 b ase pairs were readily achieved. Typically, 4 . 10(6) theoretical plat es/m were obtained in less than 30 min. Short separation times under 2 min were also possible, using the DNA intercalating dye, ethidium bro mide, along with high electric fields. The high resolving power of lin ear polyacrylamide was demonstrated in the separation of two fragments which differ by a single base pair (123/124 base pairs) using 6%T LPA and ethidium bromide intercalation. This LPA composition allowed for the possible single base-pair resolution of dsDNA fragments up to 300 base pairs in length. Several concentrations of the linear polyacrylam ide for different-ranges of fragment lengths have been employed. In ad dition, replaceable LPA offers the advantage of a fresh separation mat rix for each run, thus overcoming column stability problems and minimi zing needs for sample cleanup. Electroosmotic flow was substantially r educed using stable capillary coatings, which were required for obtain ing high efficiencies and good reproducibility.