Thesis
Cloning may not be a necessary criterion for the authentications of ancient DNA consensus sequences
Washington State University
Master of Science (MS), Washington State University
2012
Handle:
https://hdl.handle.net/2376/103555
Abstract
The challenge of studying ancient DNA (aDNA) stems principally from the degree of postmortem DNA damage witnessed by the molecules over time. This makes ancient samples prone to contamination from exogenous sources and may lead to erroneous results. The skepticism associated with the authentication of aDNA results has lead many researchers to adopt a common methods checklist, which includes cloning aDNA amplicons. However, the process of cloning is highly variable in the literature, with the number and interpretation of clones differing greatly between researchers. A previous study (Winters et al. 2011), has shown that directly sequencing ancient mammalian mitochondrial DNA (mtDNA) will not yield a disparate consensus sequence compared with cloning. A similar study has yet to be conducted on human samples, which may be more susceptible to inaccurate sequences due to the increased risk of contamination. In addition, post mortem DNA damage may also cause nucleotide misincorporations to be amplified during the PCR reaction, which may lead to aberrant results. Given the extensive use of the hypervariable region I (HVRI) in the genetic analysis of both "modern" and ancient human mtDNA, it is necessary to characterize the rate and distribution of post mortem damage in this region. To address these issues, this study was conducted to compare both cloned and directly sequenced amplicons from ~220 - 6000 year old ancient human samples amplified from four overlapping fragments within the HVRI. A majority rules approach and the Consensus Confidence Program (CCP) were used to generate consensus sequences for each individual's cloned sequences, which were then compared to the direct sequence. A relative rate of damage was calculated for nucleotide positions across 255 cloned fragments and a comparison of expected to observed counts of substitutions was analyzed using a goodness of fit test. In no instance did the consensus of clones offer any added confirmation to the endogenous DNA sequence than did the direct sequence. The amplified region was shown to exhibit a random distribution of post mortem damage that could not be significantly correlated with damage hotspots.
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Details
- Title
- Cloning may not be a necessary criterion for the authentications of ancient DNA consensus sequences
- Creators
- Misa N. Winters
- Contributors
- Brian Matthew Kemp (Degree Supervisor)
- Awarding Institution
- Washington State University
- Academic Unit
- Biological Sciences, School of
- Theses and Dissertations
- Master of Science (MS), Washington State University
- Publisher
- Washington State University; [Pullman, Washington] :
- Identifiers
- 99900525138701842
- Language
- English
- Resource Type
- Thesis