dc.contributor.author |
Vidergar, Nina |
en |
dc.contributor.author |
Toplak, Nata a |
en |
dc.contributor.author |
Kuntner, Matja |
en |
dc.date.accessioned |
2015-04-20T15:15:41Z |
|
dc.date.available |
2015-04-20T15:15:41Z |
|
dc.date.issued |
2014 |
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dc.identifier.citation |
Vidergar, Nina, Toplak, Nataša, and Kuntner, Matjaž. 2014. "<a href="http%3A%2F%2Fwww.plosone.org%2Farticle%2FfetchObject.action%3Furi%3Dinfo%253Adoi%252F10.1371%252Fjournal.pone.0113030%26representation%3DPDF">Streamlining DNA Barcoding Protocols: Automated DNA Extraction and a New cox1 Primer in Arachnid Systematics</a>." <em>PloS One</em>. 9 (11):1–12. <a href="https://doi.org/10.1371/journal.pone.0113030">https://doi.org/10.1371/journal.pone.0113030</a> |
en |
dc.identifier.issn |
1932-6203 |
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dc.identifier.uri |
http://hdl.handle.net/10088/25450 |
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dc.identifier.uri |
http://www.plosone.org/article/fetchObject.action?uri=info%3Adoi%2F10.1371%2Fjournal.pone.0113030&representation=PDF |
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dc.description.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. METHODOLOGY: 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. |
en |
dc.relation.ispartof |
PloS One |
en |
dc.title |
Streamlining DNA Barcoding Protocols: Automated DNA Extraction and a New <I>cox1</I> Primer in Arachnid Systematics |
en |
dc.type |
Journal Article |
en |
dc.identifier.srbnumber |
133116 |
|
dc.identifier.doi |
10.1371/journal.pone.0113030 |
|
rft.jtitle |
PloS One |
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rft.volume |
9 |
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rft.issue |
11 |
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rft.spage |
1 |
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rft.epage |
12 |
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dc.description.SIUnit |
NMNH |
en |
dc.description.SIUnit |
NH-Entomology |
en |
dc.description.SIUnit |
Peer-reviewed |
en |
dc.citation.spage |
1 |
|
dc.citation.epage |
12 |
|
dc.relation.url |
http://www.plosone.org/article/fetchObject.action?uri=info%3Adoi%2F10.1371%2Fjournal.pone.0113030&representation=PDF |
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