Real-time Three-Dimensional DNA Microarrays: How Well Can We Distinguish Between Related Target Sequences?

Thesis (Ph.D.)--University of Washington, 2012DNA microarrays, due to their highly parallel nature, are in principle well suited for rapid identification of known or related microbial species, but our ability to extract meaningful information from microarray images is still at a rudimentary level. The use of DNA microarrays is currently hampered by a few key analytical and theoretical challenges. In particular, the nucleic acid sequence space to be explored can be very large, the genetic sequences of many species are very similar, and the concentrations at which the different species are present is typically not known at the time of the sample collection, which can result in complex overlapping hybridization patterns. There is much disagreement in the literature regarding the merits of different microarray data analysis approaches, as they have been tested on different experimental platforms with samples of varying complexity. Experimental validation of analysis methods is limited, and not feasible as a general strategy. Advances in microarray data analysis would accelerate the employment of the powerful DNA microarray technology, already integrated into "lab-on-a-chip" instruments, in routine clinical practice. This dissertation proposes to improve the diagnostic accuracy of microarrays and characterize their detection limits with respect to distinguishing between closely related target sequences by utilizing computational microarray modeling as a tool for design and validation of microarray data analysis methods and experimental approaches. Mass transport and binding kinetics of oligonucleotide targets in gel matrices was modeled based on current understanding of the thermodynamics of DNA stability, and the performance of gel matrix microarrays as an emerging platform was characterized. Bi-phasic behavior was demonstrated in thermal dissociation curves in a multi-component system, showing promise for distinguishing between related sequences. Finally, the utility of including mismatch probes on the array for distinguishing between related sequences was explored computationally