A new sieving matrix for DNA sequencing, genotyping and mutation detection and high-throughput genotyping with a 96-capillary array system

Capillary electrophoresis has been widely accepted as a fast separation technique in DNA analysis. In this dissertation, a new sieving matrix is described for DNA analysis, especially DNA sequencing, genetic typing and mutation detection. A high-throughput 96 capillary array electrophoresis system was also demonstrated for simultaneous multiple genotyping;We first evaluated the influence of different capillary coatings on the performance of DNA sequencing. A bare capillary was compared with a DB-wax, an FC-coated and a poly(vinylpyrrolidone) (PVP) dynamically coated capillary with PEO as sieving matrix. It was found that covalently-coated capillaries had no better performance than bare capillaries while PVP coating provided excellent and reproducible results;We also developed a new sieving matrix for DNA separation based on commercially available poly(vinylpyrrolidone). This sieving matrix has a very low viscosity and an excellent self-coating effect. Successful separations were achieved in uncoated capillaries. Sequencing of M13mp18 showed good resolution up to 500 bases in treated PVP solution;Temperature gradient capillary electrophoresis and PVP solution was applied to mutation detection. A heteroduplex sample and a homoduplex reference were injected during a pair of continuous runs. A temperature gradient of 10°C with a ramp of 0.7°C/min was swept throughout the capillary. Detection was accomplished by laser induced fluorescence detection. Mutation detection was performed by comparing the pattern changes between the homoduplex and the heteroduplex samples. High throughput, high detection rate and easy operation were achieved in this system;We further demonstrated fast and reliable genotyping based on CTTv STR system by multiple-capillary array electrophoresis. The PCR products from individuals were mixed with pooled allelic ladder as an absolute standard and coinjected from a 96-vial tray. Simultaneous one-color laser-induced fluorescence detection was achieved by using a CCD camera. The allele peaks for the unknown sample were identified by comparing the normalized peak intensities of the mixtures to those of the pooled ladder by using a straightforward algorithm. An extremely high level of confidence in matching the bands was indicated with negligible cross-talk (\u3c0.89%) between adjacent capillaries

Independent Parallel Capillary Array Separations for Rapid Second Dimension Sampling in On-Line Two-Dimensional Capillary Electrophoresis of Complex Biological Samples

Biological samples remain challenging in proteomic separations due to their complexity and large concentration dynamic range. Improvements to separation power are needed to interrogate proteomes more deeply and facilitate the advancement of biomarker discovery for personalized medicine. Current online multidimensional separations require compromise; long analysis times if the second dimension (2nd-D) must be regenerated between injections, or reduced separation efficiency if the 2nd-D is operated rapidly. Using an array of capillaries as the 2nd-D, operated in parallel, allows fast sampling of the first dimension (1st-D). This relaxes the constraints on the 2nd-D separation, allowing it to operate at optimal separation conditions that would otherwise be sacrificed for speed. This configuration allows total separation times to approximately equal to the 1st-D separation time. We have developed a novel interface that enables continuous sampling of a 1st-D separation by a 2nd-D capillary array for rapid, high peak capacity two dimensional (2D) separations, based upon automated precision positioning of capillaries. Within a laminar flow regime, a capillary electrophoresis (CE) 1st-D separation was coupled to an array of eight independent CE 2nd-D separations. The instrument terminus provides laser induced fluorescence detection via a sheath flow cuvette. Effluent transfer efficiency, from the 1st-D to the 2nd-D, and detection was optimized using visible and fluorescent dye tracers. To that end, this dissertation will discuss characterization of interface and detector parameters, including: inter capillary transverse alignment accuracy, injection distance, injection time, hydrodynamic flow rate, density considerations, inter and intra capillary differences, signal crosstalk and laser intensity. Separation performance will further be demonstrated using model protein and serum digestates. Each dimension of the 2D instrument will be operated as a one dimensional (1D) instrument to compare against an optimized commercial 1D CE instrument. These results will be used to evaluate the quality of the separations operated in on-line 2D capillary electrophoresis-to-capillary array electrophoresis (CE×CAE) mode. A novel application of the CE×CAE design will be discussed in the spirit of resolving the long standing challenge of migration time reproducibility in CE separations

Capillary and microchip gel electrophoresis using multiplexed fluorescence detection with both time-resolved and spectral-discrimination capabilities: applications in DNA sequencing using near-infrared fluorescence

Increasing the information content obtainable from a single assay and system miniaturization has continued to be important research areas in analytical chemistry. The research presented in this dissertation involves the development of a two-color, time-resolved fluorescence microscope for the acquisition of both steady-state and time-resolved data during capillary and microchip electrophoresis. The utility of this hybrid fluorescence detector has been demonstrated by applying it to DNA sequencing applications. Coupling color discrimination with time-resolved fluorescence offers increased multiplexing capabilities because the lifetime data adds another layer of information. An optical fiber-based fluorescence microscope was constructed, which utilized fluorescence in near-IR region, greatly simplifying the hardware and allowing superior system sensitivity. Time-resolved data was processed using electronics configured in a time-correlated single photon counting format. Cross-talk between color channels was successfully eliminated by utilizing the intrinsic time-resolved capability associated with the detector. The two-color, time-resolved microscope was first coupled to a single capillary and carried out two-color, two-lifetime sequencing of an M13 template, achieving a read length of 650 bps at a calling accuracy of 95.1%. The feasibility of using this microscope with microchips (glass-based chips) for sequencing was then demonstrated. Results from capillaries and microchips were compared, with the microchips providing faster analysis and adequate electrophoretic performance. Lifetimes of a set of fluorescent dyes were determined with favorable precision, in spite of the low loading levels associated with the microchips. The sequencing products were required to be purified and concentrated prior to electrophoretic sorting to improve data quality. PMMA-based microchips for DNA sequencing application were evaluated. The microchips were produced from thermo plastics, which allowed rapid and inexpensive production of microstructures with high aspect ratios. It was concluded that surface coating was needed on the polymer chips in order to achieve single-base resolution required for DNA sequencing. The capability of the two-color time-resolved microscope operated in a scanning mode was further explored. The successful construction of the scanner allows scanning of multi-channel microchips for high throughput processing

Analysis of near-infrared dye-labeled Sanger sequencing fragments with gel electrophoresis using the time-resolved flourescence lifetime indentification methods

The research presented in this dissertation involves the identification of sequencing fragments with time-resolved methods. For this application, near-infrared heavy-atom tricarbocyanine dyes were developed in our laboratory, which can be excited with a single laser and emission collected using a single detection channel. The dyes have four spectroscopically unique, but relatively short lifetimes that can be altered by the intramolecular heavy-atom they contain. The work described here involves the optimization of dye-primer chemistry for preparing Sanger sequencing reactions for longer reads and the optimization of the separation matrix for capillary gel electrophoresis that produces favorable statistical analysis of the aforementioned dyes’ lifetimes. The performance of a two-lifetime experiment in which we modified an automated DNA sequencer to allow implementation of lifetime identification of DNA fragments labeled with near-IR fluorochromes and fractionation via slab-gel electrophoresis was investigated. A two-dye/two-lane sequencing experiment was carried out, in which two terminal bases, labeled with near-infrared dyes, were run in one lane and the other two bases in an adjacent lane. A lifetime evaluation of the resulting electropherogram on a pixel-by-pixel basis allowed the identification of the terminal nucleotide comprising a DNA band. The read accuracy was found to be better than a one-dye/four-lane approach using the software of the commercial instrument in spite of the fact that a spectroscopic call was implemented. An automated peak recognition and base calling algorithm was also implemented and evaluated on two-tract dye-primer and dye-terminator capillary electrophoresis runs. The base calling accuracy was greater than 97% for both

Applications of capillary electrophoresis and laser-induced fluorescence detection to the analysis of trace species: from single cells to single molecules

Several separation and detection schemes for the analysis of small volume and amount of samples, such as intracellular components and single enzymes, were developed in this work. Laser-induced fluorescence (LIF) detection provides a very sensitive approach for both direct and indirect detection in capillary electrophoresis (CE);First, indirect LIF detection and capillary electrophoresis were used to quantify lactate and pyruvate in single red blood cells. By choosing a highly efficient fluorophore and adding 1% glucose to the running buffer to stabilize the system, a detection limit of around 20 attomoles was achieved for small anions, which resulted in the easy quantification of targeted anions in single erythrocytes;The measurement of the activity for sub-attomole enzymes inside single red blood cells presents a high challenge. The assay of specific enzyme activities was achieved by monitoring the highly fluorescent enzymatic reaction product, NADH. By adding proper non-fluorescent substrates into the running buffer, the enzymes will catalyze one specific reaction after they are separated into different zones and the CE flow is stopped. The fluorescent products were related to enzyme activity. Consequently, the enzyme activity can be quantified by monitoring the fluorescent product. At about biological pH 7.4, lactate dehydrogenase (LDH) isoenzyme activities were assayed for single red blood cells. A detection limit of 1.3 x 10[superscript]-21 moles for lactate dehydrogenase was achieved by the combination of on-capillary reaction and electrophoresis. The present approach is also applicable to the assay of multiple enzymes by introducing appropriate substrates. Since lactate dehydrogenase activity serves as a good marker for certain diseases, the ability to quantify individual isoenzymes at the single cell level is of clinical importance. Leukemia cells were analyzed to evaluate the value of LDH activity as a marker for the diagnosis of leukemia. From the single cell analysis, we found that LDH activity is not a unique marker for diagnosis of leukemia, although the LDH activity in leukemia cells is lower than that in normal white blood cells;Reactions of single LDH-1 molecules were investigated by monitoring the reaction product with LIF detection. By filling a narrow capillary tube with a very low concentration of LDH-1 and excess lactate and NAD[superscript]+, discrete product zones of NADH associated with individual LDH-1 molecules are formed. We can quantify molecular concentrations down to 10[superscript]-17 M, and can also measure their activities. From the products formed during two consecutive incubation periods, each LDH-1 molecule maintains the same distinct activity over a 2-hour period. We found that the same kind of enzyme molecules can have different activities, which vary in a factor of 4. The differences in activity might be caused by different stable conformation of LDH-1 enzymes

[Capillary electrophoresis and immobilization strategies research]

During the past few decades, the development of chromatography and electrophoresis has been an essential factor for the significant advancements achieved in biotechnology. Today, efforts continue to improve upon the accuracy, speed, and precision of these methods. DNA analysis by capillary electrophoresis (CE) is a good example of how the best attributes of different methods can be brought together to develop analytical methodology that offers significant improvements over existing technology. Despite the many attributes of CE, method validation continues to be problematic. In order to reproducibly achieve high efficiency and good resolution of DNA fragments, deactivating the surface of the separation column is essential. There exist many variations to the original method first suggested in 1985 by Stellan Hjerten. In this work, scanning electron microscopy (SEM) was utilized to examine various columns coated with non cross-linked polyacrylamide. At very low concentrations of acrylamide (~2.5%), no noticeable polymer layer is present. However, as the concentration of acrylamide exceeds 2.5%, a noticeable thickness and non-uniformity is observed. The use of coated columns can then be employed for the size-, selective capillary electrophoresis (SSCE) separations of DNA fragments. Since the development of this method in the early 1990’s, several papers have discussed the theoretical aspects of utilizing aqueous solutions of soluble polymers for the separation of DNA fragments. However, the instrumentation required to directly evaluate fundamental processes such as variance in SSCE has been limited by the lack of novel instrumentation necessary to perform these experiments. In this work, experimental measurements of variance under static and dynamic conditions are reported. The determination of static diffusion coefficients and their contribution to total band variance is reported. The fact that diffusion accounts for less than half of the total variance observed led to the conclusion that other processes occurring during DNA fragments separations (i.e., DNA - polymer entanglement/disentanglement interactions) contribute significantly to band variance. Upon optimizing conditions for DNA analyses by SSCE, a novel class of cyanine intercalation dyes reported to offer superior fluorescence sensitivity relative to ethidium bromide was also evaluated in this work. Despite an improvement in sensitivity of DNA/dye complexes when employing the cyanine intercalation dyes, the labeling mechanisms and kinetics proved to be problematic in achieving appreciably lower detection limits by CE. In another area of research, the potential of utilizing modestly selective stationary phases on microsensors was evaluated. Phases commonly employed in gas chromatography (GC) and liquid chromatography (LC) were bonded onto prepared silicon substrates. The relative affinity and selectivity of these phases for semivolatile organic compounds was determined by exposing these “sensors” to solutions followed by analysis by gas chromatography/mass spectrometry (GC/MS). It was found that improving wettability of the substrate prior to phase deposition was essential to achieve uniform films. Although the relative affinity and selectivity of these films are modest, these phases may be suitable for part of a higher-order, generalized approach to sensing