title: DNA analysis of molluscs from a museum wet collection: a comparison of different extraction methods.
abstract: BACKGROUND
  DNA isolation and PCR amplification from molluscan taxa is considered as problematic because polysaccharides in tissue and mucus presumably co-precipitate with the DNA and inhibit the activity of DNA polymerase. In the present study we tested two common extraction methods on specimens from the mollusc collection of the Natural History Museum Vienna (NHMW). We analysed a broad variety of taxa covering a large temporal span (acquisition years 1877 to 1999), which distinguishes our study from previous ones where mostly fresh material was used. We also took other factors into account: effects of sample age, effects of formaldehyde treatment and taxon-specific problems. We used several primer combinations to amplify amplicons of different lengths of two mitochondrial genes: cytochrome c oxidase subunit 1 (COI) and 16S rRNA gene (16S).
  
  
  RESULTS
  Overall PCR success was 43 % in the 576 extractions (including all primer combinations). The smallest amplicon (~240 bp) showed the best results (49 % positive reactions), followed by the 400 bp amplicon (40.5 %). Both short sections yielded significantly better results than the 700 bp long amplicon (27 %). Comparatively, the Gen-ial-First, All-tissue DNA-Kit-extraction method performed significantly better than Promega-Tissue and Hair Extraction Kit. Generally, PCR success is age-dependent. Nonetheless, we were able to obtain the longest amplicon even from 137-year-old material. Importantly, formaldehyde traces did not totally inhibit amplification success, although very high concentrations did.
  
  
  CONCLUSIONS
  Museum material has gained importance for DNA analysis in recent years, especially for DNA barcoding projects. In some cases, however, the amplification of the standard barcoding region (partial sequence of the COI) is problematic with old material. Our study clearly shows that the COI barcoding region could be amplified in up to 49 % of PCRs (varying with amplicon length), which is, for museum samples, quite a high percentage. The difference between extraction methods was minimal and we recommend using an established kit for a first attempt because experience and routine in handling might be more important than slight performance differences of the various kits. Finally, we identify fixation, storage, sample conservation and documentation of the specimens' history rather than the DNA extraction method to be the most crucial factors for PCR success.
authors.affiliation: Central Research Laboratories, Natural History Museum Vienna, Burgring 7, 1010, Vienna, Austria. katharina.jaksch@nhm-wien.ac.at.|Department of Integrative Zoology, University of Vienna, Althanstraße 14, 1090, Vienna, Austria. katharina.jaksch@nhm-wien.ac.at.
authors.forename: Katharina
authors.identifier: http://orcid.org/0000-0003-2915-9878
authors.initials: K
authors.lastname: Jaksch
authors.affiliation: Third Zoological Department, Natural History Museum Vienna, Burgring 7, 1010, Vienna, Austria.
authors.forename: Anita
authors.initials: A
authors.lastname: Eschner
authors.affiliation: Leibniz Institute for Research on Evolution and Biodiversity, Museum für Naturkunde, Invalidenstraße 43, 10115, Berlin, Germany.
authors.forename: Thomas V
authors.initials: TV
authors.lastname: Rintelen
authors.affiliation: Central Research Laboratories, Natural History Museum Vienna, Burgring 7, 1010, Vienna, Austria.|Department of Integrative Zoology, University of Vienna, Althanstraße 14, 1090, Vienna, Austria.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Haring
mesh_terms: D000818:Animals; D001317:Austria; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D058893:DNA Barcoding, Taxonomic / Q000592:standards*; D017931:DNA Primers / Q000737:chemistry; D003576:Electron Transport Complex IV / Q000235:genetics; D005557:Formaldehyde / Q000737:chemistry; D059627:Liquid Phase Microextraction / Q000379:methods; D008974:Mollusca / Q000145:classification / Q000235:genetics*; D009144:Museums; D016133:Polymerase Chain Reaction / Q000592:standards*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011933:Reagent Kits, Diagnostic / Q000592:standards*; D052617:Solid Phase Microextraction / Q000379:methods; D014015:Tissue Banks
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S; D011933:Reagent Kits, Diagnostic; D005557:Formaldehyde; D004247:DNA; D003576:Electron Transport Complex IV
keywords: Ancient DNA; DNA extraction methods; Ethanol preserved specimens; Molluscs; Mucopolysaccharides; Museum material