| Summary: | Sequence based enrichment of nucleic acids is a critical enabling component of future nucleic acid detection methods in many fields including detection of nucleic acid tumor biomarkers in body fluids, non-invasive prenatal detection of fetal genetic abnormalities, and detection of pathogenic microorganisms. In many cases the problem of detecting the nucleic acid biomarker of interest is confounded by the presence of a large excess of nucleic acid sequences that may differ from the sequence of interest by only a single base. Consequently, existing methods are limited in sensitivity and amount of starting material to avoid overwhelming the detection methods with background nucleic acids. This limits their usefulness to a small number of applications. Techniques for enrichment of specific sequences rely on hybridization, and are generally not capable of enriching for low abundance sequences by more than 10 fold, a limit imposed by the thermodynamics of hybridization.
In this dissertation I present a technique for sequence enrichment of nucleic acids based on synchronous coefficient of drag alteration (SCODA), which enables sequence specific enrichment of nucleic acids from sample volumes greater than 100 μL, with concurrent concentration of the nucleic acids to volumes appropriate for PCR detection (<10 μL). We have demonstrated that this technique is capable of at least 10,000 fold enrichment of target sequences with respect to contaminating sequences differing by a single base. We have additionally shown that this technique is capable of at least 100 fold enrichment of a target sequence with a single methylated cytosine residue in a background of unmethylated targets of identical sequence by exploiting the small difference in binding energy of a methylated target to its complementary probe compared to an unmethylated target. To our knowledge this is the most specific hybridization based sequence enrichment scheme in existence, and this is the first demonstration of hybridization based enrichment of unmodified methylated DNA. Although some technical challenges must be overcome before this method will become a tool appropriate for routine laboratory use, we believe that the challenges are not insurmountable, and this method has the potential to enable routine analysis of low abundance nucleic acids.
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