Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA

Boessenkool, Sanne; Epp, Laura S.; Haile, James; Bellemain, Eva; Edwards, Mary; Coissac, Eric; Willerslev, Eske; Brochmann, Christian

Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA

Dateien

Zu diesem Dokument gibt es keine Dateien.

Datum

2012

Autor:innen

Boessenkool, Sanne

Epp, Laura S.

Haile, James

Bellemain, Eva

Edwards, Mary

Coissac, Eric

Willerslev, Eske

Brochmann, Christian

DOI (zitierfähiger Link)

10.1111/j.1365-294X.2011.05306.x

Sammlungen

Biologie

Publikationstyp

Zeitschriftenartikel

Publikationsstatus

Published

Erschienen in

Molecular ecology ; 21 (2012), 8. - S. 1806-1815. - ISSN 0962-1083. - eISSN 1365-294X

Zusammenfassung

Analyses of degraded DNA are typically hampered by contamination, especially when employing universal primers such as commonly used in environmental DNA studies. In addition to false-positive results, the amplification of contaminant DNA may cause false-negative results because of competition, or bias, during the PCR. In this study, we test the utility of human-specific blocking primers in mammal diversity analyses of ancient permafrost samples from Siberia. Using quantitative PCR (qPCR) on human and mammoth DNA, we first optimized the design and concentration of blocking primer in the PCR. Subsequently, 454 pyrosequencing of ancient permafrost samples amplified with and without the addition of blocking primer revealed that DNA sequences from a diversity of mammalian representatives of the Beringian megafauna were retrieved only when the blocking primer was added to the PCR. Notably, we observe the first retrieval of woolly rhinoceros (Coelodonta antiquitatis) DNA from ancient permafrost cores. In contrast, reactions without blocking primer resulted in complete dominance by human DNA sequences. These results demonstrate that in ancient environmental analyses, the PCR can be biased towards the amplification of contaminant sequences to such an extent that retrieval of the endogenous DNA is severely restricted. The application of blocking primers is a promising tool to avoid this bias and can greatly enhance the quantity and the diversity of the endogenous DNA sequences that are amplified.

Fachgebiet (DDC)

570 Biowissenschaften, Biologie

Schlagwörter

blocking primer, contamination, environmental DNA, metabarcoding, PCR bias, pyrosequencing

Zitieren

ISO 690

BOESSENKOOL, Sanne, Laura S. EPP, James HAILE, Eva BELLEMAIN, Mary EDWARDS, Eric COISSAC, Eske WILLERSLEV, Christian BROCHMANN, 2012. Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA. In: Molecular ecology. 21(8), pp. 1806-1815. ISSN 0962-1083. eISSN 1365-294X. Available under: doi: 10.1111/j.1365-294X.2011.05306.x

BibTex

@article{Boessenkool2012-04Block-44971,
  year={2012},
  doi={10.1111/j.1365-294X.2011.05306.x},
  title={Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA},
  number={8},
  volume={21},
  issn={0962-1083},
  journal={Molecular ecology},
  pages={1806--1815},
  author={Boessenkool, Sanne and Epp, Laura S. and Haile, James and Bellemain, Eva and Edwards, Mary and Coissac, Eric and Willerslev, Eske and Brochmann, Christian}
}

RDF

<rdf:RDF
    xmlns:dcterms="http://purl.org/dc/terms/"
    xmlns:dc="http://purl.org/dc/elements/1.1/"
    xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#"
    xmlns:bibo="http://purl.org/ontology/bibo/"
    xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#"
    xmlns:foaf="http://xmlns.com/foaf/0.1/"
    xmlns:void="http://rdfs.org/ns/void#"
    xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > 
  <rdf:Description rdf:about="https://kops.uni-konstanz.de/server/rdf/resource/123456789/44971">
    <dc:creator>Epp, Laura S.</dc:creator>
    <dc:creator>Coissac, Eric</dc:creator>
    <foaf:homepage rdf:resource="http://localhost:8080/"/>
    <dc:contributor>Willerslev, Eske</dc:contributor>
    <dc:contributor>Brochmann, Christian</dc:contributor>
    <dcterms:title>Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA</dcterms:title>
    <dc:creator>Bellemain, Eva</dc:creator>
    <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2019-02-12T10:07:36Z</dcterms:available>
    <dc:contributor>Epp, Laura S.</dc:contributor>
    <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2019-02-12T10:07:36Z</dc:date>
    <dc:contributor>Edwards, Mary</dc:contributor>
    <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/>
    <dc:language>eng</dc:language>
    <dc:contributor>Coissac, Eric</dc:contributor>
    <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/>
    <dc:contributor>Boessenkool, Sanne</dc:contributor>
    <dc:creator>Edwards, Mary</dc:creator>
    <bibo:uri rdf:resource="https://kops.uni-konstanz.de/handle/123456789/44971"/>
    <dc:creator>Haile, James</dc:creator>
    <dc:contributor>Bellemain, Eva</dc:contributor>
    <dc:creator>Brochmann, Christian</dc:creator>
    <dcterms:abstract xml:lang="eng">Analyses of degraded DNA are typically hampered by contamination, especially when employing universal primers such as commonly used in environmental DNA studies. In addition to false-positive results, the amplification of contaminant DNA may cause false-negative results because of competition, or bias, during the PCR. In this study, we test the utility of human-specific blocking primers in mammal diversity analyses of ancient permafrost samples from Siberia. Using quantitative PCR (qPCR) on human and mammoth DNA, we first optimized the design and concentration of blocking primer in the PCR. Subsequently, 454 pyrosequencing of ancient permafrost samples amplified with and without the addition of blocking primer revealed that DNA sequences from a diversity of mammalian representatives of the Beringian megafauna were retrieved only when the blocking primer was added to the PCR. Notably, we observe the first retrieval of woolly rhinoceros (Coelodonta antiquitatis) DNA from ancient permafrost cores. In contrast, reactions without blocking primer resulted in complete dominance by human DNA sequences. These results demonstrate that in ancient environmental analyses, the PCR can be biased towards the amplification of contaminant sequences to such an extent that retrieval of the endogenous DNA is severely restricted. The application of blocking primers is a promising tool to avoid this bias and can greatly enhance the quantity and the diversity of the endogenous DNA sequences that are amplified.</dcterms:abstract>
    <dc:creator>Willerslev, Eske</dc:creator>
    <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/>
    <dc:creator>Boessenkool, Sanne</dc:creator>
    <dc:contributor>Haile, James</dc:contributor>
    <dcterms:issued>2012-04</dcterms:issued>
  </rdf:Description>
</rdf:RDF>

Universitätsbibliographie

Nein

Begutachtet

Ja