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  • 1
    Language: English
    In: PLoS ONE, 2010, Vol.5(9), p.e13042
    Description: PCR amplification of minute quantities of degraded DNA for ancient DNA research, forensic analyses, wildlife studies and ultrasensitive diagnostics is often hampered by contamination problems. The extent of these problems is inversely related to DNA concentration and target fragment size and concern (i) sample contamination, (ii) laboratory surface contamination, (iii) carry-over contamination, and (iv) contamination of reagents. ; Here we performed a quantitative evaluation of current decontamination methods for these last three sources of contamination, and developed a new procedure to eliminate contaminating DNA contained in PCR reagents. We observed that most current decontamination methods are either not efficient enough to degrade short contaminating DNA molecules, rendered inefficient by the reagents themselves, or interfere with the PCR when used at doses high enough to eliminate these molecules. We also show that efficient reagent decontamination can be achieved by using a combination of treatments adapted to different reagent categories. Our procedure involves γ- and UV-irradiation and treatment with a mutant recombinant heat-labile double-strand specific DNase from the Antarctic shrimp . Optimal performance of these treatments is achieved in narrow experimental conditions that have been precisely analyzed and defined herein. ; There is not a single decontamination method valid for all possible contamination sources occurring in PCR reagents and in the molecular biology laboratory and most common decontamination methods are not efficient enough to decontaminate short DNA fragments of low concentration. We developed a versatile multistrategy decontamination procedure for PCR reagents. We demonstrate that this procedure allows efficient reagent decontamination while preserving the efficiency of PCR amplification of minute quantities of DNA.
    Keywords: Research Article ; Genetics And Genomics ; Molecular Biology ; Evolutionary Biology -- Genomics ; Molecular Biology -- Molecular Evolution
    E-ISSN: 1932-6203
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  • 2
    Language: English
    In: PLoS ONE, 01 January 2017, Vol.12(4), p.e0174216
    Description: Taxonomic over-splitting of extinct or endangered taxa, due to an incomplete knowledge of both skeletal morphological variability and the geographical ranges of past populations, continues to confuse the link between isolated extant populations and their ancestors. This is particularly problematic with the genus Equus. To more reliably determine the evolution and phylogeographic history of the endangered Asiatic wild ass, we studied the genetic diversity and inter-relationships of both extinct and extant populations over the last 100,000 years, including samples throughout its previous range from Western Europe to Southwest and East Asia. Using 229 bp of the mitochondrial hypervariable region, an approach which allowed the inclusion of information from extremely poorly preserved ancient samples, we classify all non-African wild asses into eleven clades that show a clear phylogeographic structure revealing their phylogenetic history. This study places the extinct European wild ass, E. hydruntinus, the phylogeny of which has been debated since the end of the 19th century, into its phylogenetic context within the Asiatic wild asses and reveals recent mitochondrial introgression between populations currently regarded as separate species. The phylogeographic organization of clades resulting from these efforts can be used not only to improve future taxonomic determination of a poorly characterized group of equids, but also to identify historic ranges, interbreeding events between various populations, and the impact of ancient climatic changes. In addition, appropriately placing extant relict populations into a broader phylogeographic and genetic context can better inform ongoing conservation strategies for this highly-endangered species.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 3
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 08 January 2007
    Description: Despite the enormous potential of analyses of ancient DNA for phylogeographic studies of past populations, the impact these analyses, most of which are performed with fossil samples from natural history museum collections, has been limited...
    Keywords: Life Sciences ; Biochemistry, Molecular Biology ; Molecular Biology ; Humanities and Social Sciences ; Archaeology and Prehistory ; Ancient DNA ; Bone Diagenesis ; Conservation ; DNA Conservation ; Sciences (General)
    ISSN: 0027-8424
    E-ISSN: 1091-6490
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  • 4
    Language: English
    In: Palaeogeography, Palaeoclimatology, Palaeoecology, 2008, Vol.266(3), pp.211-219
    Description: Palaeogenetic data obtained from fossilizing or fossil bones and teeth are of great importance to studies of vertebrate evolution, human biological and cultural evolution, plant and animal domestication and reconstructions of palaeoenvironment and palaeoecology. These studies are based on the retrieval of DNA preserved in fossilizing bones and teeth. DNA is present in fossils, if at all, in only very small amounts, which makes its amplification with PCR necessary for detailed sequence analysis. Erroneous nucleotides can be incorporated during amplification either because of post-mortem base damage of the original DNA template or simply because the fidelity of DNA polymerases is not absolute and can be decreased by suboptimal buffer conditions or possibly by compounds in the fossil extracts. These erroneously introduced nucleotides can be mistaken for authentic mutations of the ancient sequence compared to the closest extant sequence. Moreover, contamination by modern DNA, which is not chemically modified and therefore a better substrate for the Taq polymerase, can also lead to erroneous results. Here, we will present the procedures that we have developed in order to (i) ensure negligible mutagenicity of the PCR reaction, (ii) eliminate contamination by DNA molecules originating from previous PCR reactions and cloning procedures, (iii) prevent contamination with modern DNA of fossil bones and teeth during and after their excavation, and (iv) prevent degradation of ancient DNA after excavation. Finally, we will discuss our results on DNA preservation as a function of the taphonomy of the skeletal part that is analyzed and of the depositional context of preservation.
    Keywords: Geology
    ISSN: 0031-0182
    E-ISSN: 1872-616X
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  • 5
    Language: English
    Description: Taxonomic over-splitting of extinct or endangered taxa, due to an incomplete knowledge of both skeletal morphological variability and the geographical ranges of past populations, continues to confuse the link between isolated extant populations and their ancestors. This is particularly problematic with the genus Equus. To more reliably determine the evolution and phylogeographic history of the endangered Asiatic wild ass, we studied the genetic diversity and inter-relationships of both extinct and extant populations over the last 100,000 years, including samples throughout its previous range from Western Europe to Southwest and East Asia. Using 229 bp of the mitochondrial hypervariable region, an approach which allowed the inclusion of information from extremely poorly preserved ancient samples, we classify all non-African wild asses into eleven clades that show a clear phylogeographic structure revealing their phylogenetic history. This study places the extinct European wild ass, E. hydruntinus, the phylogeny of which has been debated since the end of the 19th century, into its phylogenetic context within the Asiatic wild asses and reveals recent mitochondrial introgression between populations currently regarded as separate species. The phylogeographic organization of clades resulting from these efforts can be used not only to improve future taxonomic determination of a poorly characterized group of equids, but also to identify historic ranges, interbreeding events between various populations, and the impact of ancient climatic changes. In addition, appropriately placing extant relict populations into a broader phylogeographic and genetic context can better inform ongoing conservation strategies for this highly-endangered species.
    Source: Open Access LMU (Universitätsbibliothek der LMU München)
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