Capillary zone electrophoresis of large DNA.

Capillary zone electrophoresis (CZE) of DNA 23.1 to 48.5 kb in length in polyacrylamide solutions of several concentrations provides evidence for polymer concentration and DNA length-dependent stretching and orientation of these species and suggests an effective separation at a polymer concentration of about 0.6%. Applying a 0.1% polyacrylamide concentration to the lambda-phage DNA ladder, at least 5 components are separated; separation improves with lowering of the field strength to 2 V/cm and, correspondingly, extended duration of CZE. Saccharomyces pombe chromosomal DNA separates into 3 major components on CZE at high field strength (270 V/cm) in 0.9% polyacrylamide solution, confirming a previous finding made on electrophoresis in a 1.1 mm ID tube at low field strength. However, the finding is limited to one source of the DNA plug, and the chromosomal identity of the components remains unknown. Methodological problems in the CZE of large DNA relate to the need for extended duration of pressure injection if absorbance detection is applied, the need to define the starting zone after extended pressure injection, the need to melt and digest agarose plugs prior to loading, and related needs for thermostating of the sample chamber and for software compatible with low voltage operation.