Lobo J et al. (2013), Enhanced primers for amplification of DNA barco...

ECB-ART-50085

BMC Ecol 2013 Sep 10;13:34. doi: 10.1186/1472-6785-13-34.

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Enhanced primers for amplification of DNA barcodes from a broad range of marine metazoans.

Lobo J , Costa PM , Teixeira MA , Ferreira MS , Costa MH , Costa FO .

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BACKGROUND: Building reference libraries of DNA barcodes is relatively straightforward when specifically designed primers are available to amplify the COI-5P region from a relatively narrow taxonomic group (e.g. single class or single order). DNA barcoding marine communities have been comparatively harder to accomplish due to the broad taxonomic diversity and lack of consistently efficient primers. Although some of the so-called "universal" primers have been relatively successful, they still fail to amplify COI-5P of many marine animal groups, while displaying random success even among species within each group. Here we propose a new pair of primers designed to enhance amplification of the COI-5P region in a wide range of marine organisms. RESULTS: Amplification tests conducted on a wide range of marine animal taxa, rendered possible the first-time sequencing of DNA barcodes from eight separated phyla (Annelida, Arthropoda, Chordata, Cnidaria, Echinodermata, Mollusca, Nemertea and Platyhelminthes), comprising a total of 14 classes, 28 orders, 57 families, 68 genus and 76 species. CONCLUSIONS: These primers demonstrated to be highly cost-effective, which is of key importance for DNA barcoding procedures, such as for building comprehensive DNA barcode libraries of marine communities, where the processing of a large numbers of specimens from a wide variety of marine taxa is compulsory.

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References [+] :

Blankenship, Universal primers and PCR of gut contents to study marine invertebrate diets. 2005, Pubmed

	Figure 1. COI sequences of three exemplificative species aligned with forward and reverse primers for the amplification of COI-5P.Clibanarius albidigitus (Phylum Arthropoda), GenBank Accession AF425321; Orconectes palmeri longimanus (Phylum Arthropoda), GenBank Accession AY701214; Bathyberthella antarctica (Phylum Mollusca), GenBank Accession AY345027. LCO1490 and HCO2198 [1]; CrustDF1 and CrustDR1 (D. Steinke unpublished in [19]); CrustF1 and CrustF2 [18]; LoboF1 and LoboR1 (present study).
	Figure 2. Image an agarose gel of COI-5P PCR products amplified using the LoboF1 and LoboR1 primer pairs. A) 100 bp Ladder, B-G) Class Bivalvia (Phylum Mollusca), H-J) Class Malacostraca (Phylum Arthropoda), K) Class Polyplacophora (Phylum Mollusca), L) Class Ophiuroidea (Phylum Echinodermata), M) Class Asteroidea (Phylum Echinodermata), N) Class Malacostraca (Phylum Arthropoda), O) Negative control. H) Possible excess of template blocked PCR amplification.
	Figure 3. Image of an agarose gel (1.5%) showing PCR products of crustaceans obtained with using different primer pairs. A), B) and F)Pilumnus hirtellus; C) and E)Lophozozymus incisus; D)Porcellana platycheles; G) Negative control; Fo) LCO1490 and HCO2198 [1]; Cr) CrustDF1 and CrustDR1 (D. Steinke unpublished in [19]); Lo) LoboF1 and LoboR1 (present study).