title: Streamlining DNA barcoding protocols: automated DNA extraction and a new cox1 primer in arachnid systematics.
abstract: BACKGROUND
  DNA barcoding is a popular tool in taxonomic and phylogenetic studies, but for most animal lineages protocols for obtaining the barcoding sequences--mitochondrial cytochrome C oxidase subunit I (cox1 AKA CO1)--are not standardized. Our aim was to explore an optimal strategy for arachnids, focusing on the species-richest lineage, spiders by (1) improving an automated DNA extraction protocol, (2) testing the performance of commonly used primer combinations, and (3) developing a new cox1 primer suitable for more efficient alignment and phylogenetic analyses.
  
  
  METHODS
  We used exemplars of 15 species from all major spider clades, processed a range of spider tissues of varying size and quality, optimized genomic DNA extraction using the MagMAX Express magnetic particle processor-an automated high throughput DNA extraction system-and tested cox1 amplification protocols emphasizing the standard barcoding region using ten routinely employed primer pairs.
  
  
  RESULTS
  The best results were obtained with the commonly used Folmer primers (LCO1490/HCO2198) that capture the standard barcode region, and with the C1-J-2183/C1-N-2776 primer pair that amplifies its extension. However, C1-J-2183 is designed too close to HCO2198 for well-interpreted, continuous sequence data, and in practice the resulting sequences from the two primer pairs rarely overlap. We therefore designed a new forward primer C1-J-2123 60 base pairs upstream of the C1-J-2183 binding site. The success rate of this new primer (93%) matched that of C1-J-2183.
  
  
  CONCLUSIONS
  The use of C1-J-2123 allows full, indel-free overlap of sequences obtained with the standard Folmer primers and with C1-J-2123 primer pair. Our preliminary tests suggest that in addition to spiders, C1-J-2123 will also perform in other arachnids and several other invertebrates. We provide optimal PCR protocols for these primer sets, and recommend using them for systematic efforts beyond DNA barcoding.
authors.affiliation: Institute of Biology, Scientific Research Centre of the Slovenian Academy of Sciences and Arts, Ljubljana, Slovenia; Molecular Virology lab, International Centre for Genetic Engineering and Biotechnology-ICGEB, Trieste, Italy.
authors.forename: Nina
authors.initials: N
authors.lastname: Vidergar
authors.affiliation: Omega d.o.o., Ljubljana, Slovenia.
authors.forename: Nataša
authors.initials: N
authors.lastname: Toplak
authors.affiliation: Institute of Biology, Scientific Research Centre of the Slovenian Academy of Sciences and Arts, Ljubljana, Slovenia; Centre for Behavioural Ecology & Evolution, College of Life Sciences, Hubei University, Wuhan, China; National Museum of Natural History, Smithsonian Institution, Washington, DC, United States of America.
authors.forename: Matjaž
authors.initials: M
authors.lastname: Kuntner
mesh_terms: D000818:Animals; D001097:Arachnida / Q000145:classification / Q000235:genetics*; D001483:Base Sequence; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003576:Electron Transport Complex IV / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012689:Sequence Homology, Nucleic Acid; D013112:Spiders / Q000145:classification / Q000235:genetics
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial; D003576:Electron Transport Complex IV