ANALYSIS OF DNA RESTRICTION FRAGMENTS AND POLYMERASE CHAIN-REACTION PRODUCTS BY CAPILLARY ELECTROPHORESIS

Capillary electrophoresis (CE) is a new, high-resolution tool for the analysis of DNA restriction fragments and DNA amplified by the polymer ase chain reaction (PCR). By combining many of the principles of tradi tional slab gel methods in a capillary format, it is possible to perfo rm molecular size determinations of human and plant PCR amplification products and DNA restriction fragments. DNA restriction fragments and PCR products were analyzed by dynamic sieving electrophoresis (DSE) an d capillary gel electrophoresis (CGE). AS part of this study, sample p reparation procedures, injection modes, and the use of molecular mass markers were evaluated. Optimum separations were performed using the u Page-3 (3% T, 3% C) CGE columns with UV detection at 260 nm. Membrane dialysis and ultrafiltration/centrifugation proved to be nearly equiva lent methods of sample preparation. Reproducibility studies demonstrat ed that blunt-ended, non-phosphorylated markers (specifically allele g enerated markers) provide the most accurate calibration for PCR produc t analysis. This study demonstrates that CE offers a high-speed, high- resolution analytical method for accurately determining molecular size and/or allelic type as compared with traditional methodologies.