On-the-fly fluorescence lifetime detection in capillary electrophoresis for mutation detection and genomic analysis

Three projects related to on-the-fly fluorescence lifetime detection (OFLD) in genomic analysis are presented in this work. The first project is the analysis of DNA point mutations and the selection of dye labels for genomic analysis. Single base anomalies in DNA sequences are often precursors or markers for genetic diseases. In this research, OFLD in capillary electrophoresis (CE) was investigated as an alternative to traditional fluorescence detection in order to improve resolution of overlapping peaks in an electropherogram.;Single base mutations in a model system were analyzed by single strand conformation polymorphism (SSCP) analysis using a variety of two-dye combinations. SSCP experiments are performed by the amplification and labeling of specific DNA base regions suspected to contain a mutation. Single stranded DNA fragments of the same length with different base sequences will migrate differentially under non-denaturing electrophoretic conditions. The dye labels studied for multiplex SSCP-OFLD analyses included 6-carboxyfluorescein (6-FAM), rhodamine green (RG), 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid (NBD), and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY FL). BODIPY FL and RG were chosen among the dyes as a suitable two-dye pair for mutation analysis because of their similar quantum yields and minimal effect on single stranded DNA mobility. This dye pair, and the pair of BODIPY FL and NBD, was used to study the G20210A prothrombin mutation. Three "blind" analyses were performed in which a control sequence along with two additional unknown sequences was analyzed, and the prothrombin mutation was correctly identified within each sample mixture.;The second project is the investigation of a design alternative for the previously developed OFLD-CE interface to enable temperature controlled analyses. Previously, the interface existed of a capillary being extended from a commercially available CE instrument to a multiharmonic Fourier transform (MHF) spectrofluorometer sample chamber. In the modified design, optical fibers were employed for the transmittance of phase-modulated excitation and emission between the CE instrument and MHF sample chamber. Fluorescence lifetimes of micromolar concentrations of fluorescein, NBD, and N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza- s-indacene-2-yl)iodoacetamide (BODIPY 507/545 IA) solutions were successfully resolved using OFLD with the modified interface. (Abstract shortened by UMI.)...