title: Collecting in collections: a PCR strategy and primer set for DNA barcoding of decades-old dried museum specimens.
abstract: Natural history museums are vastly underutilized as a source of material for DNA analysis because of perceptions about the limitations of DNA degradation in older specimens. Despite very few exceptions, most DNA barcoding projects, which aim to obtain sequence data from all species, generally use specimens collected specifically for that purpose, instead of the wealth of identified material in museums, constrained by the lack of suitable PCR methods. Any techniques that extend the utility of museum specimens for DNA analysis therefore are highly valuable. This study first tested the effects of specimen age and PCR amplicon size on PCR success rates in pinned insect specimens, then developed a PCR primer set and amplification strategy allowing greatly increased utilization of older museum specimens for DNA barcoding. PCR success rates compare favourably with the few published studies utilizing similar aged specimens, and this new strategy has the advantage of being easily automated for high-throughput laboratory workflows. The strategy uses hemi-nested, degenerate, M13-tailed PCR primers to amplify two overlapping amplicons, using two PCRs per amplicon (i.e. four PCRs per DNA sample). Initial PCR products are reamplified using an internal primer and a M13 primer. Together the two PCR amplicons yield 559 bp of the COI gene from Coleoptera, Lepidoptera, Diptera, Hemiptera, Odonata and presumably also other insects. BARCODE standard-compliant data were recovered from 67% (56 of 84) of specimens up to 25 years old, and 51% (102 of 197) of specimens up to 55 years old. Given the time, cost and specialist expertise required for fieldwork and identification, 'collecting in collections' is a viable alternative allowing researchers to capitalize on the knowledge captured by curation work in decades past.
authors: Australian Museum Research Institute, Australian Museum, 6 College Street, Sydney, NSW, 2010, Australia.
authors: Andrew
authors: A
authors: Mitchell
mesh_terms: D000818:Animals; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D017931:DNA Primers / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics; D003576:Electron Transport Complex IV / Q000235:genetics; D007313:Insecta / Q000145:classification* / Q000235:genetics*; D008969:Molecular Sequence Data; D009144:Museums; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial; D003576:Electron Transport Complex IV
keywords: COI; DNA barcodes; DNA sequencing; insect