Doctor of Philosophy (PhD)
The capacity to selectively target DNA sequences within a complex mixture is a useful feature for genomic studies. Several methods aimed at undertaking this feat have utilized biotinylated oligonucleotides and streptavidin beads to capture DNA but have low efficiencies, require PCR, or cannot be combined with high-throughput sequencing. The work presented here developed a protocol referred to as selected enrichment through capture (SEC) that uses a biotinylated “capture primer” to target and concentrate specific DNA sequences at high efficiencies, eliminates the use of PCR, and can be combined with high-throughput sequencing. The effectiveness of SEC was evaluated in a series of studies that determined the efficiency, specificity, and recovery of specific sequences from DNA mixtures. Initially, it was established that SEC could retrieve a single locus as large as 6,200 bases from 3 ng DNA with high yield. This justified attempts to retrieve 16S rRNA genes from DNA from an artificial mock community and DNA extracted from the environment. Combining SEC with high-throughput sequencing allowed for the identification of all 20 species within an artificial community, reconstruction of full-length 16S rRNA genes, and predictions of relative abundances within an order of magnitude of reported values. SEC successful captured 16S rRNA genes from bacteria from seawater, sediment, and in the over abundances of eukaryotic DNA that facilitated the characterization of the microbiome of the Eastern oyster, C. virginica. SEC was further demonstrated to be successful in recovering 16S rRNA genes from the low biomass environment of glacial basal ice with 21 pg/uL of starting DNA. Additionally, SEC was successful in retrieving insertions in the eukaryote Chlamydomonas reinhardtii. SEC has three advantages over other enrichment methods. 1) SEC does not rely on the PCR to enrich samples prior to sequencing. 2) Only a short segment of sequence adjacent to one side of the targeted region needs to be known allowing for adjacent uncharacterized sequence to be recovered. 3) Sequences of interest can be isolated, concentrated, and analyzed without PCR amplification or interference from more abundant DNA found in the sample.
Document Availability at the Time of Submission
Release the entire work immediately for access worldwide.
Nunez, Kelley Gwin, "Selective Enrichment Through Capture (SEC): A Technology for Retrieving Specific DNA Sequences from Complex Mixtures" (2015). LSU Doctoral Dissertations. 514.