File Name: dna barcodes methods and protocols .zip
Sara M. Handy and Jonathan R. Deeds U. Natalia V. Ivanova and Paul D. Andrea Ormos and Lee A. Michelle M. Moore U. Rosalee S. Hellberg U. Haile F. Yancy U. A detailed single laboratory validated protocol is provided for DNA sequencing of the cytochrome c oxidase subunit I gene COI of seafood tissue samples for the purpose of obtaining unique, species-specific "barcodes" for species identification. This LIB is no longer available online.
For additional information, contact Dr. Jonathan Deeds. Seafood is one of the most highly traded commodities in the world. In the interest of public health, it is vital that both domestically processed and imported seafood is safe, wholesome, and properly labeled. To aid in the proper labeling of seafood, the Food and Drug Administration FDA maintains a list of acceptable market names for seafood sold in U.
Organized in a series of species "pages," the RFE contains high resolution images of whole fish and their marketed product forms e. An example of an identification method listed in the RFE is protein identification by isoelectric focusing 2.
Isoelectric focusing is a currently accepted tool employed in the identification of fish fillets for regulatory compliance, but such analysis requires subjective interpretations of gel results and the inclusion of perishable frozen tissue standards in each run.
Further, the technique is not effective in the case of processed or cooked samples. The RFE was designed so that it could be expanded to include additional data and to accommodate the use of newer analytical tools as they became available. Work is furthest advanced for members of the animal kingdom. In this case, a region of the cytochrome c oxidase subunit 1 gene COI has been targeted and pilot studies have shown its effectiveness in species identification.
The Fish Barcode of Life campaign FISH-BOL is a collaborative international research effort which seeks to establish a reference library of DNA barcodes for all fish species derived from voucher specimens with authoritative taxonomic identifications 4. Fishes comprise nearly half of all vertebrate species; the group includes approximately 15, marine and 13, freshwater species FishBase. In , this protocol was evaluated by three laboratories in an inter-laboratory trial and the results of this trial were used to further refine the method.
A step by step protocol based on this published SLV study, with minor modifications, is provided below. NOTE: Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration. The process of generating DNA barcodes suitable for species identification from an unknown fish tissue sample can be broken down into the following steps:.
Individual labs will likely modify this SOP based on available equipment and reagents. In addition, for steps such as tissue lysis and DNA extraction, several commercial kits are available that may be acceptable. Any deviations from the described SOP will need to be proven to provide acceptable results. Performance criteria for individual steps are provided to aid in this process. A detailed SOP based on the single laboratory validation study detailed in Handy et al. Note: Only necessary if a subset is needed for long term storage, e.
Goal : Sample unknown fish tissues in a manner that prevents cross-contamination between samples, or the introduction of foreign DNA. Criteria for Success : After sequencing, have a chromatogram that gives single peaks for each base as evidenced by the AB1 file. Each fish or fish fillet sampled should be bagged and labeled separately.
Tissue samples should be removed with a scalpel and forceps that are flame sterilized after each sample is processed. New sterile tubes should be used for each sample. For long term storage of critical samples e. Each set of reactions also requires two negative controls, one which consists of the PCR cocktail with no additional DNA template added and another with an addition of the extraction negative control.
These samples have to be negative on the agarose gel no band produced. Goal : To produce an amplicon free of extra dNTPs and excess primers that might interfere with the sequencing reaction. Criteria for Success : Examine the PCR thermocycler to ensure that the run has completed at the correct temperatures and there are no error messages. Criteria for success : Examine the thermocycler to ensure that the run has completed at the correct temperatures and there are no error messages.
Goal : To remove unincorporated dye terminators and salts from sequencing reactions so that they will not interfere with the base pair determination of the fragment.
Several websites exist that are devoted to the troubleshooting of DNA sequencing. This is a helpful example from Etonbio. Criteria for Success : Generation of an AB1 file suitable for post sequencing analysis, though determination of the accurate base pair composition cannot be assessed until successful completion of Step 8: Post-Sequencing Analysis.
Goal : To process the sequence from the AB1 file into a usable unit for comparison with sequences from a database of authenticated standards. This database will be updated as more standards are added to the library. The database will be dated to distinguish it from previous versions. Analysts should check periodically to assure they are using the current database. In the event that a sequence from an unknown tissue sample does not match any sequence in the FDA database, the sequence can be screened against other publically available sources such as GenBank and BOLD.
These identifications should be considered presumptive unless the sequences were derived from specimens with proper authoritative taxonomic identification.
FDA will only make regulatory decisions based on identifications using adequately authenticated standards. Inclusion of sequences from publically available databases should be noted in the analytical worksheet. More elaborate statistical methods to define species boundaries are also available Kerr et al.
For example, a character-based approach was recently used to successfully distinguish some of the closely related commercial species of tuna Lowenstein et al. In some cases they can still be distinguished with this method, in other cases they cannot.
Few examples exist in the FDA Seafood List where 2 closely related species that cannot be distinguished with this method have different approved market names. Introduction Seafood is one of the most highly traded commodities in the world. Martin, Collette, and Slavin, eds. Official Method AOAC 63 ; corr.
Food Prot. Fish Biol. It is no longer available. A single laboratory validated method for the generation of DNA barcodes for the identification of fish for regulatory compliance. Modifications from Handy et al. A Nalgene 2. Protocol for Tissue Sampling Goal : Sample unknown fish tissues in a manner that prevents cross-contamination between samples, or the introduction of foreign DNA. Several tissues are suitable for DNA extraction from fishes.
These include: Musculature preferred : remove one or more cubes mm of lateral muscle skin removed. Fin clips: remove fin rays and membrane from the pectoral or pelvic fin. Use these tissues only if the fish needs to be kept alive. Eye: remove the right eye from extremely small specimens such as larvae. Buffer AL and ethanol can be premixed and added together in one step to save time when processing multiple samples.
Mark the bottle to indicate that ethanol has been added. Buffer AL can be purchased separately if the same kit will be used for purification of DNA from animal blood. Buffer AL is stable for 1 year after the addition of ethanol when stored closed at room temperature. Vortexing should be performed by pulse-vortexing for s. Include a negative extraction control with each set of extractions. The negative extraction control is treated exactly the same as sample tubes except that it does not contain any fish tissue.
Vortex occasionally during incubation to disperse the sample, or place in a thermomixer, shaking water bath, or on a rocking platform. Lysis time varies depending on the type of tissue processed. Lysis is usually complete in h.
If it is more convenient, samples can be lysed overnight; this will not affect them adversely. After incubation the lysate may appear viscous, but should not be gelatinous as it may clog the DNeasy Mini spin column. If the lysate appears very gelatinous, see the "Troubleshooting Guide", page 47, for recommendations.
Vortex for 15 s. Add Then add It is essential that the sample, Buffer AL, and ethanol are mixed immediately and thoroughly by vortexing or pipetting to yield a homogeneous solution. Note : A white precipitate may form on addition of Buffer AL and ethanol.
Their relatively slow rates of molecular evolution, as well as frequent exposure to hybridization and introgression, often make it difficult to discriminate species of vascular plants with the standard barcode markers rbcL , matK , ITS2. Previous studies have examined these constraints in narrow geographic or taxonomic contexts, but the present investigation expands analysis to consider the performance of these gene regions in discriminating the species in local floras at sites across Canada. Using plant lists from 27 national parks and one scientific reserve, we tested the efficacy of DNA barcodes in identifying the plants in simulated species assemblages from six biogeographic regions of Canada using BLAST and mothur. Mean pairwise distance MPD and mean nearest taxon distance MNTD were strong predictors of barcode performance for different plant families and genera, and both metrics supported ITS2 as possessing the highest genetic diversity. Our results indicate that DNA barcoding is very effective in identifying Canadian plants to a genus, and that it performs well in discriminating species in regions where floristic diversity is highest. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: Complete taxonomic information, collection records, voucher images and sequences for 17, specimens are publically available through BOLD Ratnasingham and Hebert in the plants of Canada project Available as of January 4, ; doi: dx.
DNA barcoding is a method of species identification using a short section of DNA from a specific gene or genes. The premise of DNA barcoding is that, by comparison with a reference library of such DNA sections also called " sequences " , an individual sequence can be used to uniquely identify an organism to species, in the same way that a supermarket scanner uses the familiar black stripes of the UPC barcode to identify an item in its stock against its reference database. Different gene regions are used to identify the different organismal groups using barcoding. The 16S rRNA gene for example is widely used in identification of prokaryotes, whereas the 18S rRNA gene is mostly used for detecting microbial eukaryotes. These gene regions are chosen because they have less intraspecific within species variation than interspecific between species variation, which is known as the "Barcoding Gap". Some applications of DNA barcoding include: identifying plant leaves even when flowers or fruits are not available; identifying pollen collected on the bodies of pollinating animals; identifying insect larvae which may have fewer diagnostic characters than adults; or investigating the diet of an animal based on its stomach content, saliva or feces.
Paul D. Hebert, T. DNA barcoding is a novel system designed to provide rapid, accurate, and automatable species identifications by using short, standardized gene regions as internal species tags. As a consequence, it will make the Linnaean taxonomic system more accessible, with benefits to ecologists, conservationists, and the diversity of agencies charged with the control of pests, invasive species, and food safety. More broadly, DNA barcoding allows a day to be envisioned when every curious mind, from professional biologists to schoolchildren, will have easy access to the names and biological attributes of any species on the planet.
Introduction. Front Matter. Pages PDF · DNA Barcodes: Methods and Protocols. W. John Kress, David L. Erickson. Pages PDF.
U.S. Food and Drug Administration
A DNA barcode in its simplest definition is one or more short gene sequences taken from a standardized portion of the genome that is used to identify species through reference to DNA sequence libraries or databases. These methods include the latest information on techniques for generating, applying, and analyzing DNA barcodes across the Tree of Life including animals, fungi, protists, algae, and plants. Thorough and intuitive, DNA Barcodes: Methods and Protocols aids scientists in continuing to study methods from wet-lab protocols, statistical, and ecological analyses along with guides to future, large-scale collections campaigns. Skip to main content Skip to table of contents.
Imagine getting bitten by a spider, but being unable to tell what kind of spider it was poisonous or not?! To help organize our understanding of the diversity of species in the living world, Carl Linneaus invented a system for naming and classifying organisms in We still use this system today, and call it taxonomy. In Linnaean taxonomy, all the different kinds of living organisms can be organized practically into groupings with shared characteristics, where every species can be given a unique name. Biologists often identify species based on the way they look anatomy , behavior, or evolutionary history, and assign them to a taxonomic grouping.
Four species of Paphiopedilum were sampled and barcoded.
DNA Barcodes: Methods and Protocols
DNA Barcodes pp Cite as. DNA barcoding, a new method for the quick identification of any species based on extracting a DNA sequence from a tiny tissue sample of any organism, is now being applied to taxa across the tree of life. As a research tool for taxonomists, DNA barcoding assists in identification by expanding the ability to diagnose species by including all life history stages of an organism. As a biodiversity discovery tool, DNA barcoding helps to flag species that are potentially new to science.
Sara M. Handy and Jonathan R. Deeds U. Natalia V.