Abstract
BackgroundBuilding 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.ResultsAmplification 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.ConclusionsThese 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.
Highlights
Building reference libraries of DNA barcodes is relatively straightforward when designed primers are available to amplify the COI-5P region from a relatively narrow taxonomic group
The universal primers designed by Folmer and colleagues [1] (HCO2198LCO1490, named “Folmer primers”) for amplification of a 658 base pair fragment of the 5′ end of the mitochondrial gene cytochrome c oxidase subunit I (COI-5P), have shown to be very successful in the amplification of this gene fragment in a broad range of marine metazoan phyla
The primers LoboF1 and LoboR1 amplified the COI-5P (Figure 2) from a wide diversity of species from different animal phyla (Table 1), both vertebrates and invertebrates, comprising 130 specimens belonging to 76 species from 8 phyla
Summary
Building reference libraries of DNA barcodes is relatively straightforward when designed primers are available to amplify the COI-5P region from a relatively narrow taxonomic group (e.g. single class or single order). The universal primers designed by Folmer and colleagues [1] (HCO2198LCO1490, named “Folmer primers”) for amplification of a 658 base pair (bp) fragment of the 5′ end of the mitochondrial gene cytochrome c oxidase subunit I (COI-5P), have shown to be very successful in the amplification of this gene fragment in a broad range of marine metazoan phyla. HCO2198 primer pair, but the efforts were unsuccessful, as they were with different combinations of primers. In another example, Blankenship et al [7], failed to amplify COI-5P using the Folmer primers from the remnants of big-eye tuna (identified only through the mitochondrial 16S gene) inside the guts of deep-sea amphipods (Scopelocheirus schellenbergi). Given the vast geographic distribution and the important commercial value of holothurians, further work should focus on developing alternative primers for these species, because PCR (polymerase chain reaction) amplification could not be achieved with the primers available [11]
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