Separation and detection of nucleic acids

This dissertation investigates novel methodologies for the separation and detection of nucleic acids. The ability of pulsed field gel electrophoresis (PFGE) to resolve large single stranded DNA fragments is first examined. A methodology is presented to systematically separate electric field, temperature and pulsing effects during PFGE experiments. The influence of these parameters on DNA migration is evaluated and no particular pulsing effect is found to be operative for DNA fragments in this size range. The method outlined is expected to be of general utility when investigating other pulsed field geometries.;The utility of matrix assisted laser desorption/ionization (MALDI) mass spectrometry as a sequencing technique is examined as well. A methodology is described to purify and mass analyze Sanger sequencing reactions using the bacteriophage M13 as template DNA. The result demonstrates feasibility of the approach, but a loss of signal is observed for fragments longer than 35 base pairs. This effect is ascribed to DNA fragmentation, itself dependent on the analyte sequence and the matrix used. Investigations of matrix influence are, however, complicated by the inhomogeneous and irreproducible nature of the MALDI samples. To facilitate matrix studies, a new sample preparation is presented which utilizes a self-assembled monolayer of matrix. This method replaces the conventional matrix/analyte crystals and offers uniform and homogeneous desorption media for model studies of the MALDI processes.;Electrospray ionization mass spectrometry (ESI-MS) allows analysis of DNA without fragmentation. However, multiple charging of the analytes yields complex mass spectra and prevents analysis of DNA sequencing mixtures. A charge reduction method using Polonium as a neutralizer is investigated in order to form singly charged ESI ions. Feasibility of the approach is demonstrated in ESI mass spectrometry and differential mobility analysis (ESI-DMA). The charge reduction effects are evaluated as functions of the ion source geometry and running parameters, and a new ion source design is proposed to achieve complete neutralization in ESI-MS. The utility of ESI-DMA for nucleic acid analysis is evaluated with single and double stranded fragments.