Software Tools for Design of Reagents for Multiplex Genetic Analyses

Abstract: Methods using oligonucleotide probes are powerful tools for the analysis of nucleic acids. During recent years, many such methods have been developed that enable the simultaneous interrogation of multiple qualities of a sample. Many of these multiplexing techniques share common limitations. This thesis discusses new developments to overcome the problems of multiplex amplification of genomic sequences and design of sets of oligonucleotide probes for multiplex genetic analyses.A novel molecular technique, termed the selector method, is described. This method allows circularization of an arbitrary selection of restriction fragments from genomic DNA, and the subsequent amplification of these circular products in parallel using common primers. The utility of the method is demonstrated by a 96-plex amplification experiment. Furthermore, the PieceMaker software for selection of restriction enzymes and restriction fragments is described. These two developments allow the selective amplification of subsets of genomes for further analyses.A software tool for the design of sequence-tagged oligonucleotide probes is presented. The ProbeMaker software is a framework for design of sets of probes composed of separate functional elements, and uses an extension mechanism to incorporate support for new probe types as needed. An approach to a unified system for oligonucleotide design is also presented. This system will serve to decrease development times for new oligonucleotide design applications by allowing extensive code reuse.