DNA separations by free-solution conjugate electrophoresis in capillaries and on microchips for sequencing and genotyping applications [electronic resource]
Keywords: Chemical Engineering

Capillary electrophoresis in a viscous sieving polymer was used to sequence the first human genome by separating DNA molecules on basis of size. Since the completion of the Human Genome Project in 2003, several "next-generation" technologies have offered greatly decreased cost per base and increased throughput of DNA sequencing. While the ultra-high-throughput nature of these technologies is highly advantageous to genome-wide studies, it is not amenable to all situations; electrophoresis-based sequencing remains relevant when a small number of exons need to be sequenced rapidly and accurately. Miniaturized electrophoretic separations on microfluidic devices require less sample volume, a shorter analysis time, and, in principle, can be performed on an inexpensive, disposable platform. The high pressures necessary to load polymer solutions into chips are challenging and difficult to automate. This work presents a method to separate DNA by size with no polymer matrix to enable rapid, accurate DNA sequencing and size-based genotyping on microchips, with an approach that can be automated and/or multiplexed. Free-Solution Conjugate Electrophoresis (FSCE) achieves size-based separations of DNA by virtue of the attachment of a mobility-modifying "drag-tag" to the end of DNA molecules. Drag-tags must be completely monodisperse so that only one bioconjugate peak appears in the electropherogram for each length of DNA. Previous work in the Barron lab focused on the development of highly repetitive polypeptide "protein polymer" drag-tags, since chemically synthesized polymers which were sufficiently large, while also being monodisperse, are not available. However, the Barron lab struggled for seven years to produce recombinant proteins with sufficient length and monodispersity to enable DNA sequencing. This dissertation presents significant improvements to the development and use of protein polymer drag-tags for size-based separations of DNA. The cause of the polydispersity previously plaguing the production and purification of protein polymer drag-tags was discovered and eliminated. A longer, monodisperse protein polymer of the previously designed repetitive "family" enabled an almost 50% increase in read length by capillary electrophoresis, which is the longest FSCE sequencing read ever reported, and is essentially "on par" with the read lengths of current next-generation technologies. The longest drag-tag produced is predicted to be able to sequence at least 400 bases of DNA. A second "family" of repetitive protein polymers was also developed, which allows the addition of positively charged amino acid residues for increased friction without extra length to be tested. For all protein drag-tags, interactions between the slightly positively charged drag-tags and microchannel walls were found to cause increased peak width when more than 3 cationic amino acids were present, despite the use of robust and hydrophilic dynamic wall coatings. Significant work was done to transition FSCE separations of DNA oligomers conjugated to drag-tags onto glass microfluidic devices. An optimal injection method for separations in buffer was discovered, and all the monodisperse drag-tags were able to be injected when conjugated to ssDNA oligomers, including the largest protein (516 amino acids long). Separation efficiency by microchip electrophoresis was tested. Contrary to matrix-based separations, free-solution electrophoresis with drag-tags shows no loss of resolution when electric field strength is increased, up to the maximum of the power supply (E = 700 V/cm). This indicates that the speed of free-solution sequencing separations will be able to be minimized while still achieving single-base resolution, all without the use of a polymeric sieving network. Specific recommendations are made for further research advances necessary to implement FSCE sequencing separations on microchips. In addition to separating DNA sequencing fragments, FSCE was applied to size-based separation of DNA molecules for genotyping using the ligase detection reaction (LDR). Initial proof-of-concept experiments and further multiplexing demonstrated the advantages of FSCE genotyping on plastic and glass microchips. Using four drag-tags, FSCE-LDR was used to identify, simultaneously, all 19 mutant loci in the K-ras gene of diagnostic importance to colorectal cancer in less than 75 seconds on a glass microchip.