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

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2012
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Boessenkool, Sanne
Haile, James
Bellemain, Eva
Edwards, Mary
Coissac, Eric
Willerslev, Eske
Brochmann, Christian
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Molecular ecology. 2012, 21(8), pp. 1806-1815. ISSN 0962-1083. eISSN 1365-294X. Available under: doi: 10.1111/j.1365-294X.2011.05306.x
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.

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570 Biowissenschaften, Biologie
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blocking primer, contamination, environmental DNA, metabarcoding, PCR bias, pyrosequencing
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ISO 690BOESSENKOOL, 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. 2012, 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}
}
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