Optimization of DNA Recovery and Amplification from Non-Carbonized Archaeobotanical Remains
Open accessAbstract
Ancient DNA (aDNA) recovered from archaeobotanical remains can provide key insights into many prominent archaeological research questions, including processes of domestication, past subsistence strategies, and human interactions with the environment. However, it is often difficult to isolate aDNA from ancient plant materials, and furthermore, such DNA extracts frequently contain inhibitory substances that preclude successful PCR amplification. In the age of high-throughput sequencing, this problem is even more significant because each additional endogenous aDNA molecule improves analytical resolution. Therefore, in this paper, we compare a variety of DNA extraction techniques on primarily desiccated archaeobotanical remains and identify which method consistently yields the greatest amount of purified DNA. In addition, we test five DNA polymerases to determine how well they replicate DNA extracted from non-charred ancient plant remains. Based upon the criteria of resistance to enzymatic inhibition, behavior in quantitative real-time PCR, replication fidelity, and compatibility with aDNA damage, we conclude these polymerases have nuanced properties, requiring researchers to make educated decisions as to which one to use for a given task. The experimental findings should prove useful to the aDNA and archaeological communities by guiding future research methodologies and ensuring precious archaeobotanical remains are studied in optimal ways, and may thereby yield important new perspectives on the interactions between humans and past plant communities.
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Previous studies have reported lower error rates and higher extension termination rates for Pfu-based polymerases (such as Phusion) compared to Taq-based and other uracil-tolerant polymerases.
“Previous studies have reported lower error rates and higher extension termination rates for Pfu‐based polymerases (such as Phusion) compared to Taq‐based and other uracil‐tolerant polymerases (Heyn et al., 2010; Ginolhac et al., 2011; Wales et al., 2014).”
From Successful enrichment and recovery of whole mitochondrial genomes from ancient human dental calculus (2016) · cites this paper as Wales et al., 2014
The Results (DNA Polymerases, Fidelity) report significantly lower substitution error rates for Phusion than for the other tested polymerases. The Results (Compatibility with damaged DNA) and Discussion say Phusion stalls at uracil in damaged templates while the others read through it, but the paper does not report comparative extension-termination rates. It also identifies PfuTurbo Cx as a uracil-tolerant comparator, so the finding should not be generalized to all Pfu-based polymerases. (LLM, conf=0.85)
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- Authors
- Wales, N., Andersen, K., Cappellini, E., Ávila-Arcos, M. C., & Gilbert, M. T. P.
- Affiliations
- University of Copenhagen (DK)
- Corresponding author
- Wales, Nathan
- Andersen, Kenneth
- Cappellini, Enrico
- Gilbert, M. Thomas P.
- Venue
- PLoS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2014-01-27
- Volume
- 9
- Issue
- 1
- Pages
- e86827
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC3903575
- OpenAlex
- W2052673034
- Cited by
- 80
- References
- 60
- Citations by year
- 2026: 5 · 2025: 6 · 2024: 3 · 2023: 6 · 2022: 8 · 2021: 9 · 2020: 8 · 2019: 8 · 2018: 10 · 2017: 6 · 2016: 5 · 2015: 4 · 2014: 2
- FWCI
- 4.14
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- PLoS ONE (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- eSpace (Curtin University) (submittedVersion, cc-by)
- Figshare (submittedVersion, cc-by)
- Funders
- American-Scandinavian Foundation
- Arizona State University
- University of Connecticut
- National Academy of Sciences of Armenia
- National Research Foundation
- Danmarks Grundforskningsfond (DNRF94)
- Danmarks Frie Forskningsfond (10‐081390)
- Topics
- Physical Sciences › Earth and Planetary Sciences › Paleontology › Archaeology and ancient environmental studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Forensic and Genetic Research
- Physical Sciences › Environmental Science › Ecology › Environmental DNA in Biodiversity Studies
- Keywords
- ancient DNA, archaeological plant remains, DNA extraction, PCR amplification, DNA polymerase, enzyme inhibition, DNA damage, replication fidelity
- MeSH terms
- Analysis of Variance, Base Sequence, Botany, DNA-Directed DNA Polymerase, Molecular Sequence Data, Paleontology, Plants, DNA, Plant, Nucleic Acid Amplification Techniques, High-Throughput Nucleotide Sequencing
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