Quantitation of fluorescent nucleotide incorporation by capillary gel electrophoresis and laser-induced fluorescence detection.

A method has been developed by which enzymatically incorporated fluorophore-labeled nucleotide sites in nucleic acid can be quantitated by degradation of nanogram quantities of DNA followed by capillary gel electrophoretic analysis with fluorescence detection. In this way the differing relative labeling densities achieved using either C5-substituted dUTP's or N4-substituted dCTP's were determined. The method has proven to be very useful in obtaining quantitative analytical data from the small quantities of complex molecules produced in nick translations. Various polymerization conditions using DNA polymerase I were examined to determine optimal labeling density. Simultaneous copolymerization of green fluorescing dCTP and dUTP nucleotides were undertaken in an attempt to maximize labeling density.

Adaptation of capillary isoelectric focusing to microchannels on a glass chip.

As a first step toward adaptation of capillary isoelectric focusing (cIEF) to microchannels on a glass chip, we have compared the three most common mobilization methods: chemical, hydrodynamic, and electroosmotic flow (EOF)-driven mobilization. Using a commercial cIEF apparatus with coated or uncoated fused-silica capillaries, both chemical and hydrodynamic mobilization gave superior separation efficiency and reproducibility. However, EOF-driven mobilization, which occurs simultaneously with focusing, proved most suitable for miniaturization because of high speed, EOF compatibility and low instrumentation requirements. When this method was tested in a 200-micron-wide, 10-micron-deep, and 7-cm-long channel etched into planar glass, a mixture of Cy5-labeled peptides could be focused in less than 30 s, with plate heights of 0.4 micron (410 plates/s) upon optimization. For a total analysis time of less than 5 min, we estimate a maximum peak capacity of approximately 30-40. Interestingly, the order of migration was found to be reversed compared to capillary-based focusing.

Simultaneous multiple analyte detection using fluorescent peptides and capillary isoelectric focusing.

Analyte-specific detection based on the isoelectric point of the detection moiety is a new concept that is under investigation at Vysis. We have developed methods for the synthesis of of fluorescent synthetic peptides that can be conjugated to bioanalytes such as nucleic acids and antibodies, processed in a hybridization or binding assay, and then chemically released prior to detection by capillary isoelectric focusing (cIEF)-laser-induced fluorescence (LIF) detection. A two-step cIEF method in coated capillaries using salt mobilization has been used that produces high peak efficiencies and good assay reproducibility. The concentration by focusing aspect of cIEF, which allows for the entire capillary to be filled with sample, enables detection limits in the pM as opposed to sub-nM level for conventional capillary electrophoresis (CE)-LIF. The simultaneous multiple detection of eleven different focusing entities has been achieved.

Characterization of N-acyl-D-biotinols by particle-beam liquid chromatography-mass spectrometry. An alternative to probe mass spectrometry for thermally labile samples.

In the course of preparing biotin-labeled nucleic acid probes, it was necessary to verify structures of intermediate N-acyl derivatives of biotinol. Characterization by mass spectrometry (MS) involved use of particle-beam liquid chromatography (LC)-mass spectrometry MS to supplement standard heated-solids probe techniques. The probe data for a sample of N-toluoylbiotinol indicated it to be a mixture of mono- and di-toluoylbiotinols which was inconsistent with other analytical information. Analysis of the same sample by LC-MS on a reversed-phase column with a water-acetonitrile gradient showed a single major peak with spectrum consistent with that for the monotoluoyl species. These results suggested that a thermal transacylation reaction might be occurring in the probe during heating prior to volatilization and ionization. This was confirmed by heating the sample to 200 degrees C and then repeating the LC-MS analysis to find peaks now present for biotinol and ditoluoylbiotinol as well as the starting material. These results demonstrate the value of particle-beam LC-MS as a technique for obtaining electron-impact mass spectra of thermally sensitive compounds.