Electrophoretic separations of polymerase chain reaction-amplified DNA fragments in DNA typing using a capillary electrophoresis- laser induced fluorescence system

Abstract

Analysis of polymerase chain reaction (PCR)-amplified DNA fragments for human identification requires high-resolution separation and efficient detection of amplified alleles. Capillary electrophoresis (CE) with its speed, automation, high resolution and efficiency shows promise for analysing the amplified DNA fragments. CE with UV detection, however, suffers from lack of detector sensitivity owing to the limited detection path length of the capillary. By the use of intercalating dyes (TOTO and YOYO) a laser-induced fluorescence (LIF) detection system can provide much greater sensitivity for detecting DNA fragments. Femtogram quantities of dsDNA (φX174 HaeIII restriction digest mixture) per nanoliter of injected volume have been detected. Application of CE-LIF to analysis of PCR-amplified DNA fragments from three different genetic loci (apolipoprotein B, VNTR locus D1S80, mitochondrial DNA) is shown here. Further, the resolving power of a polymer-network capillary separation system is compared to that of a capillary-gel separation system.