title: Comparison of Suitability of the Most Common Ancient DNA Quantification Methods.
abstract: OBJECTIVE
  Ancient DNA (aDNA) extracted from historical bones is damaged and fragmented into short segments, present in low quantity, and usually copurified with microbial DNA. A wide range of DNA quantification methods are available. The aim of this study was to compare the five most common DNA quantification methods for aDNA.
  
  
  METHODS
  Quantification methods were tested on DNA extracted from skeletal material originating from an early medieval burial site. The tested methods included ultraviolet (UV) absorbance, real-time quantitative polymerase chain reaction (qPCR) based on SYBR®green detection, real-time qPCR based on a forensic kit, quantification via fluorescent dyes bonded to DNA, and fragmentary analysis. Differences between groups were tested using a paired t-test.
  
  
  RESULTS
  Methods that measure total DNA present in the sample (NanoDrop™UV spectrophotometer and Qubit®fluorometer) showed the highest concentrations. Methods based on real-time qPCR underestimated the quantity of aDNA. The most accurate method of aDNA quantification was fragmentary analysis, which also allows DNA quantification of the desired length and is not affected by PCR inhibitors.
  
  
  CONCLUSIONS
  Methods based on the quantification of the total amount of DNA in samples are unsuitable for ancient samples as they overestimate the amount of DNA presumably due to the presence of microbial DNA. Real-time qPCR methods give undervalued results due to DNA damage and the presence of PCR inhibitors. DNA quantification methods based on fragment analysis show not only the quantity of DNA but also fragment length.
authors.affiliation: 1 Laboratory of Biological and Molecular Anthropology, Institute of Experimental Biology, Faculty of Science, Masaryk University , Brno, Czech Republic .
authors.forename: Kristýna
authors.initials: K
authors.lastname: Brzobohatá
authors.affiliation: 1 Laboratory of Biological and Molecular Anthropology, Institute of Experimental Biology, Faculty of Science, Masaryk University , Brno, Czech Republic .
authors.forename: Eva
authors.initials: E
authors.lastname: Drozdová
authors.affiliation: 2 Biovendor, Laboratory Medicine , Brno, Czech Republic .
authors.forename: Jiří
authors.initials: J
authors.lastname: Smutný
authors.affiliation: 3 Department of Anthropology, Faculty of Natural Sciences, Comenius University , Bratislava, Slovak Republic .
authors.forename: Tomáš
authors.initials: T
authors.lastname: Zeman
authors.affiliation: 3 Department of Anthropology, Faculty of Natural Sciences, Comenius University , Bratislava, Slovak Republic .
authors.forename: Radoslav
authors.initials: R
authors.lastname: Beňuš
mesh_terms: D004247:DNA / Q000235:genetics; D000072441:DNA, Ancient / Q000032:analysis* / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D000072441:DNA, Ancient; D004247:DNA
keywords: aDNA fragmentation; aDNA quantification; ancient DNA; real-time qPCR