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All Subjects: Biology

Using Ancient DNA Methods to Examine Dire Wolf Population History

Description

Dire wolves have recently risen to fame as a result of the popular television program Game of Thrones, and thus many viewers know dire wolves as the sigil and loyal companions of the Stark house. Far fewer recognize dire wolves by their scientific name, Canis dirus, or understand the population history of this ‘fearsome wolf’ species that roamed the Americas until the megafaunal mass extinction event of the Late Pleistocene. Although numerous studies have examined the species using morphological and geographical methods, thus far their results have been either inconclusive or contradictory. Remaining questions include the relationships dire wolves share with other members of the Canis genus and the internal structure of their populations. Advancements in ancient DNA recovery methods may make it possible to study dire wolf specimens at the molecular level for the first time and may therefore prove useful in clarifying the answers to these questions. Eighteen dire wolf specimens were collected from across the United States and subjected to ancient DNA extraction, library preparation, amplification and purification, bait preparation and capture, and next-generation sequencing. There was an average of 76.9 unique reads and 5.73% coverage when mapped to the Canis familiaris reference genome in ultraconserved regions of the mitochondrial genome. The results indicate that endogenous ancient DNA was not successfully recovered and perhaps ancient DNA recovery methods have not advanced to the point of retrieving informative amounts of DNA from particularly old, thermally degraded specimens. Nevertheless, the ever-changing nature of ancient DNA research makes it vital to continually test the limitations of the field and suggests that ancient DNA recovery methods will prove useful in illuminating dire wolf population history at some point in the future.

ContributorsSkerry, Katherine Marie (Author) / Stone, Anne (Thesis director) / Amdam, Gro (Committee member) / Larson, Greger (Committee member) / School of Human Evolution and Social Change (Contributor) / School of Nutrition and Health Promotion (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)

Created2018-05

Novel DNA Extraction Methods for Mollusks and the History and Significance of Bermuda Land Snails

Description

Bermuda Land Snails make up a genus called Poecilozonites that is endemic to Bermuda and is extensively present in its fossil record. These snails were also integral to the creation of the theory of punctuated equilibrium. The DNA of mollusks is difficult to sequence because of a class of proteins called mucopolysaccharides that are present in high concentrations in mollusk tissue, and are not removed with standard DNA extraction methods. They inhibit Polymerase Chain Reactions (PCRs) and interfere with Next Generation Sequencing methods. This paper will discuss the DNA extraction methods that were designed to remove the inhibitory proteins that were tested on another gastropod species (Pomacea canaliculata). These were chosen because they are invasive and while they are not pulmonates, they are similar enough to Bermuda Land Snails to reliably test extraction methods. The methods that were tested included two commercially available kits: the Qiagen Blood and Tissue Kit and the Omega Biotek Mollusc Extraction Kit, and one Hexadecyltrimethylammonium Bromide (CTAB) Extraction method that was modified for use on mollusk tissue. The Blood and Tissue kit produced some DNA, the mollusk kit produced almost none, and the CTAB Extraction Method produced the highest concentrations on average, and may prove to be the most viable option for future extractions. PCRs attempted with the extracted DNA have all failed, though it is likely due to an issue with reagents. Further spectrographic analysis of the DNA from the test extractions has shown that they were successful at removing mucopolysaccharides. When the protocol is optimized, it will be used to extract DNA from the tissue from six individuals from each of the two extant species of Bermuda Land Snails. This DNA will be used in several experiments involving Next Generation Sequencing, with the goal of assembling a variety of genome data. These data will then be used to a construct reference genome for Bermuda Land Snails. The genomes generated by this project will be used in population genetic analyses between individuals of the same species, and between individuals of different species. These analyses will then be used to aid in conservation efforts for the species.

ContributorsClark, Patrick Louis (Author) / Stone, Anne (Thesis director) / Winingear, Stevie (Committee member) / School of Life Sciences (Contributor, Contributor) / School of Human Evolution & Social Change (Contributor) / Barrett, The Honors College (Contributor)

Created2021-05

Comparison of Two DNA Extraction Methods for Isolating Mycobacterium leprae DNA from FFPE Tissue Collected in the Pacific Islands Region

Description

Mycobacterium leprae, the causative agent of Hansen’s disease (leprosy), has plagued humans and other animal species for millennia and remains of concern to public health throughout the world today. Recent research into the expanded use of medical tissues preserved as formalin-fixed, paraffin-embedded samples (FFPE), opened the door for the study of M. leprae DNA from preserved skin samples. However, problems persist with damage to the DNA including fragmentation and cross linkage. This study evaluated two methods commonly used for the recovery of host DNA from FFPE samples for their efficacy in extracting pathogen DNA (hot alkaline lysis protocol and QIAGEN QIAamp FFPE DNA kit). Twenty FFPE skin samples collected from 1995-2015 from human subjects in the Pacific Islands suffering from M. leprae infection, each exhibiting a range of bacillary loads, were analyzed to determine which extraction method was most successful in terms of ability to consistently yield reliable, robust traces of M. leprae infection. This study further examined these samples to understand the phylogeny of leprosy in the region, where gaps in the evolutionary history of M. leprae persist. DNA recovery from paired samples was similar using either method. However, by extending the incubation time of post-paraffin removal sample lysis, both protocols were more likely to yield positive traces of M. leprae, with this enhancement being especially evident in paucibacillary samples with low bacterial presence. The qPCR assay findings suggest that the hot alkaline procedure is most likely to yield positive identification of infection in these traditionally challenging samples.

ContributorsKing, Felicia Clarice (Author) / Stone, Anne (Thesis advisor) / Wilson, Melissa (Committee member) / Buetow, Ken (Committee member) / Arizona State University (Publisher)

Created2023