Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA
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 bi...
Published in: | Molecular Ecology |
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2012
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ftsouthampton:oai:eprints.soton.ac.uk:473701 2023-07-30T04:06:17+02:00 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 2012-04 https://eprints.soton.ac.uk/473701/ English eng Boessenkool, Sanne, Epp, Laura S., Haile, James, Bellemain, Eva, Edwards, Mary, Coissac, Eric, Willerslev, Eske and Brochmann, Christian (2012) Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA. Molecular Ecology, 21 (8), 1806-1815. (doi:10.1111/j.1365-294X.2011.05306.x <http://dx.doi.org/10.1111/j.1365-294X.2011.05306.x>). Article PeerReviewed 2012 ftsouthampton https://doi.org/10.1111/j.1365-294X.2011.05306.x 2023-07-09T22:57:43Z 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. Article in Journal/Newspaper permafrost Siberia University of Southampton: e-Prints Soton Molecular Ecology 21 8 1806 1815 |
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University of Southampton: e-Prints Soton |
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ftsouthampton |
language |
English |
description |
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. |
format |
Article in Journal/Newspaper |
author |
Boessenkool, Sanne Epp, Laura S. Haile, James Bellemain, Eva Edwards, Mary Coissac, Eric Willerslev, Eske Brochmann, Christian |
spellingShingle |
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 |
author_facet |
Boessenkool, Sanne Epp, Laura S. Haile, James Bellemain, Eva Edwards, Mary Coissac, Eric Willerslev, Eske Brochmann, Christian |
author_sort |
Boessenkool, Sanne |
title |
Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA |
title_short |
Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA |
title_full |
Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA |
title_fullStr |
Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA |
title_full_unstemmed |
Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA |
title_sort |
blocking human contaminant dna during pcr allows amplification of rare mammal species from sedimentary ancient dna |
publishDate |
2012 |
url |
https://eprints.soton.ac.uk/473701/ |
genre |
permafrost Siberia |
genre_facet |
permafrost Siberia |
op_relation |
Boessenkool, Sanne, Epp, Laura S., Haile, James, Bellemain, Eva, Edwards, Mary, Coissac, Eric, Willerslev, Eske and Brochmann, Christian (2012) Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA. Molecular Ecology, 21 (8), 1806-1815. (doi:10.1111/j.1365-294X.2011.05306.x <http://dx.doi.org/10.1111/j.1365-294X.2011.05306.x>). |
op_doi |
https://doi.org/10.1111/j.1365-294X.2011.05306.x |
container_title |
Molecular Ecology |
container_volume |
21 |
container_issue |
8 |
container_start_page |
1806 |
op_container_end_page |
1815 |
_version_ |
1772818794492723200 |