Development of capillary electrophoretic separation methods for PCR-amplified DNA products
This research has qualified the utility of capillary electrophoresis (CE) in DNA typing work for human identification and enabled ultra sensitive detection of PCR-amplified DNA fragments. In accomplishing the above goal, various capillaries such as polyacrylamide coated capillaries, polyacrylamide gel-filled capillaries and polysiloxane coated capillaries such as SPB-5 (5% phenyl and 95% methyl groups) and DB-17 (50% phenyl and 50% methyl groups) were evaluated and used in conjunction with several buffers and polymers such as hydroxypropylmethylcellulose, hydroxyethylcellulose and methylcellulose. These studies indicated the suitability of a coated capillary with sieving polymers (polymer network capillaries) for the analysis of PCR-amplified DNA products. The investigations of separation parameters like applied field on the separation efficiency and resolution of PCR-amplified DNA fragments showed that, in general, resolution and efficiency for the larger fragments (greater than 600 basepairs) decreased with increase in applied field. Fitting the data to current migration models confirmed the gradual transition to reptation regime concurrent with similar findings by other groups. The results of the temperature studies showed that efficiency and resolution decreased with increase in temperature, hence, all PCR-amplified dsDNA separations were performed at ambient temperature. Injection studies showed suitability of electrophoretic injection for PCR-amplified dsDNA separations. Smaller injection plugs gave higher efficiency and resolution but compromised detectability of the analyte using UV detection. Investigations on sample preparation prior to analysis by CE showed suitability of an Ultrafree-MC filter unit (Millipore Corp, MA), which had the advantage of speed and recovery. Studies on the use of modifiers like glycerol, chaotropic agents like urea and intercalating agents like ethidium bromide, showed selective improvement in separations especially for dsDNA fragments less than 300 basepairs and were found suited for separations of smaller sized PCR-amplified DNA products. Sensitivity enhancement over UV detection was achieved by the use of two asymmetric cyanine dyes, TOTO and YOYO, coupled with a laser induced fluorescence detection system (LIF). Femtogram quantities of dsDNA were detected by this approach. The resolving power of a polymer network capillary separation system when compared to that of a capillary-gel separation system, clearly confirmed the advantage of using polymer networks for PCR-amplified dsDNA analysis with a CE-LIF system. The overall goal of this project was to enable ultra sensitive detection and high resolution separation of PCR-amplified human DNA fragments from different loci. (Abstract shortened by UMI.).