Recent developments in DNA sequencing technologies have resulted in substantial reductions in time and cost for large scale genome sequencing efforts. However, human genome sequencing remains out of reach in small laboratory and clinical settings due to the massive size and complexity of our genomes. To satisfy the increasing demand for genomic sequence information for biomedical research and personalized healthcare, next-generation DNA sequencing technologies are expected to achieve unprecedented multiplexing, sample throughput and cost reductions. To realize these goals, a substantial decrease in the size of the sequencing device and the implementation of a massively parallel DNA preparation and analysis scheme will be required. In response to these needs, I am developing methods for amplifying, sorting and arraying single DNA clones to maximize throughput and imaging efficiency and minimize reagent waste associated with many DNA sequencing technologies. I am also developing an electrophoretic device that will enhance the stringency and reduce the background fluorescence associated with DNA sequencing by ligation. The utility of these methods will be demonstrated through the development of a genome-scale, high-throughput assay for quantifying transcription factor binding specificities
AIM 1: Develop a method for massively parallel clonal amplification of genomic DNA on microbeads.
The state of the art in DNA amplification utilizes the emulsion polymerase chain reaction (emPCR) process. This method uses water-in-oil micro-emulsions to isolate beads and templates so that monoclonal products can be produced on the beads. Micro-emulsions that contain both a bead and a single DNA template must occur at very low frequencies, a requirement that leads to wasted reagents and added costs. In addition, the size of the micro-emulsions is difficult to control, resulting in variation in the amount of amplified product and wasted reagents. Bead populations must also be enriched to remove the large portion of beads that do not contain any PCR products. The goal of this project is to demonstrate that massively parallel clonal amplification via PCR can be performed on similar beads in microfabricated reactors within an automated fluidics system in a more efficient, consistent, and cost-effective manner than emPCR.
AIM 2: Develop a method for fabricating wafer-scale, high-density arrays of DNA-conjugated microbeads on glass cover slips.
Some of the emerging “next-generation” sequencing platforms utilize randomly distributed DNA-conjugated microbeads or clones on a glass slide within a reaction chamber. The random arrangements of the beads or clones result in low throughput and imaging efficiency, complicated image processing and high reagent costs. One approach to dramatically improve these devices involves the use of arrays to eliminate overlap and to minimize the area between the beads or clones. Such arrays can be generated by depositing the DNA samples onto the glass slides using a printing method or by assembling beads onto microfabricated arrays of wells that have been etched into glass, silicon or the face of a fiber-optic bundle. However, for genome sequencing applications, the fabrication process needs to be scalable and inexpensive. The format of the arrays must also be compatible with high-throughput imaging and microfluidics devices. The goal of this project is to combine photolithographic techniques with the facilitated self-assembly of microbeads to create large, high-density arrays of DNA clones. For more information, see Barbee, K.D.; Huang, X. Magnetic Assembly of High-Density DNA Arrays for Genomic Analyses. Analytical Chemistry (2008)
AIM 3: Improve the speed and accuracy of DNA hybridization and massively parallel sequencing by ligation via an electrophoresis-based microbead array platform.
The accuracy of DNA sequencing by ligation (SBL) depends upon the incorporation of the correct probes and is especially critical for single molecule approaches. Nonspecific binding of probes is also a significant problem when thermal stringency alone is used during washing steps. The incorporation of an electrophoretic system within a DNA sequencing platform will enable regulated transport of oligonucleotides and greater discrimination during hybridization of the probes. The goal of this project is to construct an electrophoresis chamber that will drastically improve high-throughput SBL by enhancing the efficiency and stringency of hybridization and ligation of oligonucleotides, and by eliminating background fluorescence from nonspecifically bound oligonucleotides.
AIM 4: Develop a high-throughput method for quantifying transcription factor binding specificities on a genome scale.
The regulation of gene transcription is one of the most important mechanisms of cellular control. The transcription factors (TFs) involved in the regulation of gene expression are proteins and protein complexes that recognize and bind to the promoters and other regulatory sequences of the genes. The systematic determination of the binding specificities of TFs will be essential for the elucidation of the complex genetic regulatory networks and pathways. Technologies such as ChIP-on-chip, protein binding microarrays, and mechanically induced trapping of molecular interactions have been developed to perform such analyses. However, none of these methods has sufficient throughput for analyzing the binding sites of a large number of TFs across an entire human genome. The goal of this project is to apply our array fabrication and sequencing technologies to develop a method for studying TF binding specificities.
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