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Biological evidence may contain any number of cells in any proportion. Extreme low-template DNA samples are often very difficult to interpret due to complex signal or peaks which may be indistinguishable from baseline noise. Current solutions focus on increasing the amount of amplicon detected by adjusting PCR cycle number or capillary electrophoresis injection parameters. Consensus profiling is an additional option. However, the aforementioned solutions are often not helpful for extreme low-template samples due to the high occurrence of allelic drop-out. Additionally, PCR is a destructive technique that causes one amplification to completely exhaust this type of sample, making further typing and analysis impossible. Therefore, a technique that allows for the re-generation of a DNA template in order to amplify it multiple times would be an extremely useful tool. This study outlines the development of a method that allows for the recursive amplification of a DNA sample. Amplification was performed using biotinylated primers for an STR locus and the resulting product was cleaned using streptavidin-coated magnetic beads to sequester the amplicons. Subsequent centrifugal filtration was used to remove the remaining PCR components, thus isolating the original genomic DNA. Re-amplification was then successfully performed at a different STR locus. Though successful, multiple run-throughs of the method indicated retention of signal from the original amplification as well as significant genomic DNA loss during the process. This study outlines experiments seeking to characterize the cause(s) of these imperfections in order to effectively direct method optimization. A computer generated dynamic model was also created and used to simulate the recursive amplification process to assist in development. When optimized, it is expected that recursive amplification can significantly reduce the difficulties associated with low-template DNA analysis and eradicate the concept of an ‘exhaustive’ DNA sample.

Although DNA microarray can detect multiple DNA samples simultaneously, current detection techniques involve PCR and other traditional procedures. In this study, a sensitive, specific and rapid detection method, which eliminates PCR and other lengthy processes, for pathogenic DNA is presented. This technology is based on the hybridization of target DNA to the immobilized probe, extension of probe DNAs using the target-DNA as a template and signal generation by streptavidin-horseradish peroxidase and substrate. This method is highly specific and sensitive, allowing single-nucleotide-base mismatches discrimination and the detection at femtomole level. The experiments are designed to achieve short hybridization time. Therefore, satisfactory signal can be detected within minutes, allowing the rapid detection of multiple pathogenic DNA. Most importantly, the E. coli genomic DNA can be detected using this technology. In conclusion, this detection method is useful for applications including on-site pathogenic disease detection, crime scene investigation, and pathogen inspection in the environment.

Combinatorial approaches in metabolic engineering can make use of randomized mutations and/or overexpression of randomized DNA fragments. When DNA fragments are obtained from a common genome or metagenome and packaged into the same expression vector, this is referred to as a DNA library. Generating quality DNA libraries that incorporate broad genetic diversity is challenging, despite the availability of published protocols. In response, a novel, efficient, and reproducible technique for creating DNA libraries was created in this research based on whole genome amplification using degenerate oligonucleotide primed PCR (DOP-PCR). The approach can produce DNA libraries from nanograms of a template genome or the metagenome of multiple microbial populations. The DOP-PCR primers contain random bases, and thermodynamics of hairpin formation was used to design primers capable of binding randomly to template DNA for amplification with minimal bias. Next-generation high-throughput sequencing was used to determine the design is capable of amplifying up to 98% of template genomic DNA and consistently out-performed other DOP-PCR primers. Application of these new DOP-PCR amplified DNA libraries was demonstrated in multiple strain enrichments to isolate genetic library fragments capable of (i) increasing tolerance of E. coli ER2256 to toxic levels of 1-butanol by doubling the growth rate of the culture, (ii) redirecting metabolism to ethanol and pyruvate production (over 250% increase in yield) in Clostridium cellulolyticum when consuming cellobiose, and (iii) enhancing L-arginine production when used in conjunction with a new synthetic gene circuit. / Ph. D.