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

Actin nanokinematics under the influence of DC electric fields

Molecular shuttles are nanometer-sized machines capable of transporting single molecules over small distances under user control. Actin-myosin system is a motor protein system, which naturally evolved within the cell for the nanoscale transport. The Acto-Myosin system is fuelled by ATP, converting chemical energy into linear motion. The gliding geometry movement of actin wherein the tails of the motors are adsorbed to the surface and the heads of the motors move the actin filaments across the surface can be exploited to achieve the directional control. The motion of actin in a specific direction and the surface of travel can be modified either through lithographic methods or imprinting techniques. The aim of this thesis is to estimate and quantize the motion of Actin filaments under electric force fields. The required electrical fields are simulated and the mobility of actin under the electric force field is estimated quantitatively. Experimental exploration of this motion was targeted. (Abstract shortened by UMI.)

Investigating routes for in vitro and in vivo data storage

PhD thesisComputing Science, Synthetic Biology and Nanotechnology are converging. Synthetic Biology and Nanotechnology compose the “hardware” platform, whilst Computing Science formulates the logic, data storage and processing pipelines in order to create complex yet controlled behaviour at the nanoscale. Although much work has been done on information processing at the nanoscale via in vivo constructs, e.g. logic gates in various organisms, relatively little has been done on implementing data structure, a fundamental building block for computation. This dissertation proposes and investigates methods to implement data structures by employing biological molecules via both a Synthetic Biology and a Nanotechnological approach. A data structure implemented at the nanoscale could help to substantially increase the complexity of behaviours that could be programmed and embedded in living cells or at the interface between living cells and other nano-substrates, with potential applications in intelligent drug factories and delivery nanosystems, biosensors, and environmental cleaning bionanotechnologies. This work explores the possibility of implementing via DNA constructs, both in vitro and in vivo, "list-like" data structure that can potentially hold an unlimited number of items. This has not been achieved before. Thus, the text describes designs and test prototypes. Firstly, this thesis focuses on an in vitro approach. This is achieved through a DNA-based machinery implementing a signal recorder based on DNA strand displacement reactions. Such DNA architecture can in principle implement a stack machine, capable of storing data providing a dynamic temporary memory capable of pushing and popping data-items encoded in DNA nanostructures (called DNA "bricks”). The "list-like" data is thus represented by a growing (or shrinking) chain of DNA bricks. iv Secondly, I introduce a potential design and initial experiments for an in vivo approach presenting, a synthetic genetic circuit designed to record and accumulate extracellular signals digitally within a "tape" DNA molecule inside a living cell. The core is based on the engineering of the self-splicing group II retrotransposon Ll.LtrB of Lactococcus lactis. Together, these two in vitro and in vivo routes expand our knowledge in the context of molecular memory devices and the biological operations we can compute

Solid-phase DNA sequencing reactions performed in micro-capillary reactors and solid-phase reversible immobilization in microfluidic chips for purification of dye-labeled DNA sequencing fragments

The research presented in this dissertation involves micro-capillary reactors for solid phase DNA sequencing, the identification of dye terminator sequencing fragments with time-resolved methods, and purification of dye-labeled DNA fragments using solid- phase reversible immobilization in microfluidic chips. Solid-phase micro-reactors have been prepared for DNA sequencing applications using slab gel electrophoresis. A PCR product was immobilized to the interior wall of a fused-silica capillary tube via a biotin-streptavidin linkage. Solid-phase sequencing was carried out in micro-capillary reactors using a four-lane, single color dye primer chemistry strategy. The read length was found to be 589 bases. The complementary DNA fragments generated in the small volume (~62 nL) reactor were directly injected into the gel-filled capillary for size separation with detection accomplished using near-IR laser-induced fluorescence. A set of terminators labeled with near-IR heavy-atom modified tricarbocyanine dyes were investigated for a terminator sequencing protocol in conjunction with slab gel electrophoresis. This protocol gave 605 bp read lengths. A one color, two lifetime format of DNA sequencing was implemented. A pixel-by-pixel analysis was employed to identify each of the bases in the run. The resulting read accuracy for the two-dye capillary run was 90.6%. The use of photoactivated polycarbonate (PC) for purification of dye-labeled terminator sequencing fragments using solid-phase reversible immobilization (SPRI) was investigated. SPRI cleanup of dye-terminator sequencing fragments using a photoactivated PC microchannel and slab gel electrophoresis produce a read length of 620 bases with a calling accuracy of 98.9%. The PC-SPRI cleanup format was also integrated to a capillary gel electrophoresis system. In this case, the immobilization microchannel contained microposts to increase the loading level of DNAs to improve signal intensity without the need for pre-concentration