title: Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA.
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 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.
authors: National Centre for Biosystematics, Natural History Museum, University of Oslo, Oslo, Norway. sanneboessenkool@gmail.com
authors: Sanne
authors: S
authors: Boessenkool
authors: Laura S
authors: LS
authors: Epp
authors: James
authors: J
authors: Haile
authors: Eva
authors: E
authors: Bellemain
authors: Mary
authors: M
authors: Edwards
authors: Eric
authors: E
authors: Coissac
authors: Eske
authors: E
authors: Willerslev
authors: Christian
authors: C
authors: Brochmann
mesh_terms: D000818:Animals; D004247:DNA / Q000032:analysis / Q000302:isolation & purification; D059018:DNA Contamination; D017931:DNA Primers / Q000235:genetics*; D005580:Fossils; D019015:Geologic Sediments / Q000737:chemistry; D006801:Humans; D007053:Ice; D010527:Perissodactyla / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods; D012800:Siberia
chemical_list: D017931:DNA Primers; D007053:Ice; D004247:DNA