The University of Southampton
University of Southampton Institutional Repository

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

Data from: Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA
Data from: 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 due to 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 optimised 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 (C. 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 analysis, the PCR can be biased towards 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.,Boessenkool_Blocker_seqBoessenkool_Blocker_seq_id,
DRYAD
Boessenkool, Sanne
6a24c3b2-94e2-4722-bd5a-33041ce5e6c9
Epp, Laura S.
d7c2dd0c-c6be-4dfa-a1a8-3f76187fcc6c
Haile, James
831f077c-daae-4aa1-b4b7-c4c76a2f6c00
Bellemain, Eva
9361d0fe-4c0f-49c7-9570-92924556a212
Edwards, Mary
4b6a3389-f3a4-4933-b8fd-acdfef72200e
Coissac, Eric
66ae82c0-bd94-4446-8d1b-af0d15732d0b
Willerslev, Eske
3815b419-7ba2-4827-a7a7-e97dd7118ee6
Brochmann, Christian
82cfe10a-4506-4340-a219-8ee809f52d5e
Boessenkool, Sanne
6a24c3b2-94e2-4722-bd5a-33041ce5e6c9
Epp, Laura S.
d7c2dd0c-c6be-4dfa-a1a8-3f76187fcc6c
Haile, James
831f077c-daae-4aa1-b4b7-c4c76a2f6c00
Bellemain, Eva
9361d0fe-4c0f-49c7-9570-92924556a212
Edwards, Mary
4b6a3389-f3a4-4933-b8fd-acdfef72200e
Coissac, Eric
66ae82c0-bd94-4446-8d1b-af0d15732d0b
Willerslev, Eske
3815b419-7ba2-4827-a7a7-e97dd7118ee6
Brochmann, Christian
82cfe10a-4506-4340-a219-8ee809f52d5e

Boessenkool, Sanne, Epp, Laura S., Haile, James, Bellemain, Eva, Coissac, Eric, Willerslev, Eske and Brochmann, Christian (2011) Data from: Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA. DRYAD doi:10.5061/dryad.t33v4k14 [Dataset]

Record type: Dataset

Abstract

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 due to 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 optimised 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 (C. 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 analysis, the PCR can be biased towards 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.,Boessenkool_Blocker_seqBoessenkool_Blocker_seq_id,

Full text not available from this repository.

More information

Published date: 1 January 2011

Identifiers

Local EPrints ID: 448686
URI: http://eprints.soton.ac.uk/id/eprint/448686
PURE UUID: c8be93c8-93ff-401e-bd88-31b32921f6c0
ORCID for Mary Edwards: ORCID iD orcid.org/0000-0002-3490-6682

Catalogue record

Date deposited: 29 Apr 2021 16:32
Last modified: 30 Apr 2021 01:38

Export record

Altmetrics

Contributors

Creator: Sanne Boessenkool
Creator: Laura S. Epp
Creator: James Haile
Creator: Eva Bellemain
Contributor: Mary Edwards ORCID iD
Creator: Eric Coissac
Creator: Eske Willerslev
Creator: Christian Brochmann

University divisions

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of http://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×