Name: DNA-based Fish Species Identification Protocol (Video) | JoVE Journal
Uploaded: 2010-04-28T17:00:00.000+00:00
Duration: 9 min 15 s
Description: Learn DNA-based fish species identification using PCR and RFLP analysis with the Agilent system. This method enables accurate seafood authenticity testing for processed samples in less than one workday.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

Agilent Stratagene Products Division 
<ul>
<li>Harini Ravi</li>
<li>Natalia Novorodovskaya</li>
<li>Scott Basehore</li>
<li>Jeff Braman</li>
<li>Rachel Formosa</li>
<li>Ronda Allen</li>
<li>Rajesh Bagga</li>
<li>Derek Hall</li>
<li>Sarah Jandle</li>
<li>Danielle Huffman</li>
</ul>

Agilent Laboratories
<ul>
<li>Robert Kincaid</li>
<li>Mary McBride</li>
</ul>

Agilent Europe
<ul>
<li>Nigel Skinner</li>
<li>Juergen Schneider</li>
<li>Stephen Mueller</li>
<li>Martin Kratzmeier</li>
</ul>

Campden BRI
<ul>
<li>Steve Garrett</li></ul>

1: Prepare the Fish Samples for DNA Extraction
<ol>
<li>For each fish sample to be tested, place a piece of the fish tissue, weighing between 10 mg and 1 g (raw or cooked), into a single 1.5-ml microcentrifuge tube. Use the figure below as a guideline for estimating the weight of a raw fish sample based on sample size. 
<img src="/files/ftp_upload/1871/1871fig1.jpg" alt="Salmon sample comparison; weight vs size; scale chart; research on fish sample metrics; biology study." data-altupdatedat="1788235205000" data-alt="figure 1"> 
Figure 1. Mass Estimations of Fish Samples Based on Sample Size. </li>
<li>Pre-warm the Proteinase K Digestion Buffer to 65°C for 5 minutes in an incubator or water bath.</li>
<li>Prepare a working solution of Proteinase K by combining 200 μl of Proteinase K Digestion Buffer and 20 μl of Proteinase K per sample. 
Note: Prepare a fresh working solution of Proteinase K before each use.</li>
<li>Add 220 μl of the Proteinase K working solution to each 1.5-ml tube of fish sample. Incubate the tubes at 65°C for 10 minutes in an incubator or water bath.</li>
<li>Spin the tubes in a microcentrifuge for 3 5 minutes at 14,000 x g to pellet any undigested tissues.</li>
<li>Transfer 150 μl of each supernatant into a fresh 1.5-ml tube. Avoid transferring any undigested material from the bottom of the tube or any oily material that may be present at the top of the tube. These tubes of supernatant are the samples from which genomic DNA will be extracted.</li>
</ol>
2: Extract Genomic DNA
<ol>
<li>In a sterile container (polypropylene tube or glass bottle), prepare a working solution of sulfolane and Nucleic Acid Binding Buffer by combining 320 μl of 90% sulfolane and 170 μl of Nucleic Acid Binding Buffer per sample. 
You may want to prepare a sufficient quantity of this mixture to process as many samples as you plan to examine within the next 4 weeks. This mixture can be stored for up to 30 days at room temperature. Avoid exposing the mixture to light during storage.</li>
<li>Add 490 μl of the sulfolane/Binding Buffer mixture (prepared in step 1 above) to each fish sample. Vortex or pipet the sample repeatedly until homogenized. The addition of this mixture will bring the total volume of each sample to 640 μl.</li>
<li>Transfer each sample to a DNA Binding Spin Cup that has been seated within a 2-ml receptacle tube (provided) and snap the cap of the tube onto the top of the spin cup. </li>
<li>Spin the samples in a microcentrifuge for 1 minute at 14,000 x g to load the DNA onto the spin cup matrix. </li>
<li>Remove and retain the spin cups and discard the filtrates. For each sample, replace the spin cup in the receptacle tube, then add 500 μl of 1x High Salt Wash Buffer and cap the tube. </li>
<li>Spin the samples in a microcentrifuge at 14,000 x g for 1 minute.</li>
<li>Remove and retain the spin cups and discard the filtrates. For each sample, replace the spin cup in the receptacle tube, then add 500 μl of 80% ethanol and cap the tube.</li>
<li>Spin the samples in a microcentrifuge at 14,000 x g for 1 minute. </li>
<li>Repeat steps 7 and 8 two more times for a total of 3 washes with 500 μl of 80% ethanol. </li>
<li>After the third wash in 80% ethanol, remove and retain the spin cups and discard the filtrates. Replace the spin cups in their receptacle tubes and spin in a microcentrifuge for 2 minutes at 14,000 x g to dry the fiber matrix.</li>
<li>Transfer the spin cups to 1.5-m1 collection tubes. Add 100 μl of Elution Buffer to each spin cup directly on the fiber matrix inside the cup. Snap the caps of the collection tubes onto the spin cups and incubate at room temperature for 1 minute.</li>
<li>Spin the samples in a microcentrifuge at maximum speed for 1 minute.</li>
<li>The purified DNA is in the Elution Buffer in the microcentrifuge tube. Discard the spin cup and cap the tubes. The DNA may be stored at 4°C for up to one month. For long-term storage, store the DNA at 20°C or 80°C.</li>
<li>If desired, you may measure the concentration of the DNA samples in a spectrophotometer.</li></ol> 
The genomic DNA extraction protocol typically yields samples with a concentration ranging from 5 ng/μl to 500 ng/μl. The PCR-RFLP protocol works wells with DNA samples ranging anywhere from 0.05 ng/μl to 2000 ng/μl.
3: Set up the PCR Reactions
<ol> 
<li>	Prepare a 50 ng/μl dilution of the positive control salmon DNA by combining 8 μl of the DNA stock with 32 μl of sterile, DNase-free water. Vortex to mix. 
The diluted sample may be stored at 4°C for future use.</li> 
<li>	Prepare the reactions by combining the components in the table below in order. Prepare a single reagent mixture for all PCR reactions that will be run simultaneously by scaling up the volumes listed in the table. In addition to the test DNA samples, include a positive control reaction and a no-template control reaction. Preparing duplicate PCR reactions for each test DNA sample is recommended. Prepare enough reagent mixture for all your reactions plus one reaction volume excess. 
For example, if you have 5 test DNA samples, prepare enough reagent mixture for either 8 reactions (5 test reactions, 1 positive control, 1 no-template control, and 1 excess) or 13 reactions if you are including duplicates of the test reactions (10 duplicate test reactions, 1 positive control, 1 no-template control and 1 excess). 
PCR Reagent Mixture
<table border="1">
 <tbody>
 <tr>
 <td>Component</td>
 <td>Volume 1 Reaction</td>
 <td>Volume 5 Reactions</td>
 </tr>
 <tr>
 <td>dH2O, sterile</td>
 <td>9 μl</td>
 <td>45 μl</td>
 </tr>
 <tr>
 <td>2x PCR Master Mix</td>
 <td>12.5 μl</td>
 <td>62.5 μl</td>
 </tr>
 <tr>
 <td>Primer Mix</td>
 <td>2.5 μl</td>
 <td>12.5 μl</td>
 </tr>
 <tr>
 <td>Total volume</td>
 <td>24 μl</td>
 <td>120 μl</td>
 </tr>
 </tbody>
</table>
</li>
<li>Vortex the reagent mixture well, then distribute 24 μl to each individual thin-walled PCR reaction tube.</li>
<li>Add 1 μl of the diluted positive control DNA to the positive control reaction tube. To the test sample tubes, add 1 μl of test DNA sample. For the no-template control reaction, add 1 μl of DNase-free water in place of the DNA. To avoid cross-contamination, use a fresh pipet tip for each DNA sample. After adding the sample, mix the reaction by quickly pipetting the contents of the tube up and down.</li>
<li>Cap the reaction tubes, vortex the tubes to mix and centrifuge the tubes briefly.</li></ol>
4: Run the PCR Protocol
<ol>
<li>Place the reactions in the thermal cycler and run the PCR program shown below. 
PCR Cycling Protocol
<table border="1">
 <tbody>
 <tr>
 <td>Segment</td>
 <td>Number of Cycles</td>
 <td>Temperature</td>
 <td>Duration</td>
 </tr>
 <tr>
 <td>1</td>
 <td>1</td>
 <td>95°C</td>
 <td>5 minutes</td>
 </tr>
 <tr>
 <td>2</td>
 <td>40</td>
 <td>95°C 50°C 72°C</td>
 <td>30 seconds 30 seconds 30 seconds</td>
 </tr>
 <tr>
 <td>3</td>
 <td>1</td>
 <td>72°C</td>
 <td>7 minutes</td>
 </tr>
 </tbody>
</table> 
</li></ol>
5: Digest PCR Products with Restriction Enzymes 
<ol>
<li>Label the 0.5-ml tubes or 0.2-ml strip tubes that are to be used for the restriction digest reactions. Each PCR reaction will be digested with three different restriction enzymes: DdeI, HaeIII and NlaIII. Therefore, for each PCR reaction, label three separate tubes with the name of the PCR sample and the name of the restriction enzyme.</li>
<li>Prepare the reagent mixture for the DdeI digestions by combining the components below in order. Prepare a single reagent mixture for all DdeI digestion reactions (plus at least one reaction volume excess) using multiples of each component. Once the PCR is complete, the PCR reactions are treated with restriction enzymes for restriction fragment length polymorphism (RFLP) analysis. 
DdeI Digestion Reagent Mixture
<table border="1">
 <tbody>
 <tr>
 <td>Component</td>
 <td>Volume</td>
 </tr>
 <tr>
 <td>dH2O, sterile</td>
 <td>1.5 μl</td>
 </tr>
 <tr>
 <td>10x DdeI Buffer</td>
 <td>0.5 μl</td>
 </tr>
 <tr>
 <td>10x DdeI enzyme</td>
 <td>0.5 μl</td>
 </tr>
 </tbody>
</table>
</li>
<li>Vortex the reagent mixture well, then distribute 2.5 μl to the individual reaction tubes that were labeled for DdeI.</li>
<li>Prepare the reagent mixture for the HaeIII digestions by combining the components below in order. Prepare a single reagent mixture for all HaeIII digestion reactions (plus at least one reaction volume excess) using multiples of each component. 
HaeIII Digestion Reagent Mixture
<table border="1">
 <tbody>
 <tr>
 <td>Component</td>
 <td>Volume</td>
 </tr>
 <tr>
 <td>dH2O, sterile</td>
 <td>1.5 μl</td>
 </tr>
 <tr>
 <td>10x HaeIII Buffer</td>
 <td>0.5 μl</td>
 </tr>
 <tr>
 <td>10x HaeIII enzyme</td>
 <td>0.5 μl</td>
 </tr>
 </tbody>
</table>
</li>
<li>Vortex the reagent mixture well, then distribute 2.5 μl to the individual reaction tubes that were labeled for HaeIII.</li>
<li>Prepare the reagent mixture for the NlaIII digestions by combining the components below in order. Prepare a single reagent mixture for all NlaIII digestion reactions (plus at least one reaction volume excess) using multiples of each component. 
NlaIII Digestion Reagent Mixture
<table border="1">
 <tbody>
 <tr>
 <td>Component</td>
 <td>Volume</td>
 </tr>
 <tr>
 <td>dH2O, sterile</td>
 <td>1.5 μl</td>
 </tr>
 <tr>
 <td>10x NlaIII Buffer</td>
 <td>0.5 μl</td>
 </tr>
 <tr>
 <td>10x NlaIII enzyme</td>
 <td>0.5 μl</td>
 </tr>
 </tbody>
</table>
</li>
<li>Vortex the reagent mixture well, then distribute 2.5 μl to the individual reaction tubes that were labeled for NlaIII.</li>
<li>For each digestion reaction, add 2.5 μl of the appropriate PCR product to the labeled tubes. All of the test PCR reactions as well as the positive control reaction need to be digested with all three restriction enzymes.</li>
<li>Vortex the digestion reactions and then briefly centrifuge the tubes.</li>
<li>Incubate all the digestion reactions at 37°C for 2 hours. This incubation can be performed in the thermal cycler. If desired, reactions may be left at 37°C overnight.</li>
<li>Transfer the reactions to 65°C for 15 minutes. This step can be performed in the thermal cycler.</li>
<li>Add 1 μl of 60 mM EDTA (provided with the kit) to each reaction and vortex well.</li>
</ol>
6: Analyze the restriction digest patterns 
<ol>
<li>Prepare the gel-dye mix, place a DNA chip on the chip priming station, and load the mix onto the DNA chip.</li>
<li>Pipet 5 μl of DNA marker into the well marked with the ladder symbol and into each of the 12 sample wells on the chip. 
DNA marker is provided in the DNA 1000 Reagent Kit in a green-capped tube.</li>
<li>Pipet 1 μl of DNA ladder into the well marked with a ladder symbol. 
DNA ladder is provided in the DNA 1000 Reagent Kit in a yellow-capped tube.</li>
<li>For each of the DNA samples, pipet 1 μl of each restriction digest reaction into one of the 12 sample wells on the chip according to the guidelines in the figure below. Using this approach, wells 1-3 are for the 3 digestion reactions for the positive control sample, wells 4-6 are for the 3 digestion reactions for test sample 1, wells 7-9 are for the 3 digestion reactions for test sample 2, and wells 10-12 are for the 3 digestion reactions for test sample 3.Analyze the restriction digest reactions on the Agilent 2100 Bioanalyzer to determine the fragment lengths produced during digestion. The Agilent DNA 1000 Kit Guide has complete instructions for running the lab chips on the Bioanalyzer. A brief protocol is provided below for your convenience. 
<img src="/files/ftp_upload/1871/1871fig2.jpg" alt="DNA chip diagram for on-chip electrophoresis; test samples comparison and control sample analysis." data-altupdatedat="1788235205000" data-alt="figure 2"> 
Figure 2. Organization of Samples on Chip. 
If you have less than 4 DNA samples, pipet 1 μl of water into the unused wells. If you have more than 4 DNA samples, you will need to run more than one chip. Note that it is not necessary to run the positive control sample on every chip.</li>
<li>Place the chip horizontally in the adapter of the IKA vortex mixer and vortex for 60 seconds at 2400 rpm.</li>
<li>Insert the chip into the Agilent 2100 Bioanalyzer and start the chip run. The incoming raw signals are displayed in the Instrument context. 
After the run has finished, peaks are identified for all samples using the settings of the peak find algorithm. If you believe the algorithm has failed to detect a genuine peak, you may lower the Height Threshold setpoint to a value that allows the algorithm to identify the peak.</li>
<li>Go to the Assay context and select the Chip Summary tab. In the Sample Name field, enter a sample name for all 12 wells on the chip as shown in the figure below. 
<img src="/files/ftp_upload/1871/1871fig3.jpg" alt="gene analysis results table; PCR samples, controls listed, data entry interface" data-altupdatedat="1788235205000" data-alt="figure 3"> 
Figure 3. Sample Name Entry in Chip Summary Tab.</li></ol>
7: Identify the test sample species using RFLP Matcher 
The Agilent software application RFLP Decoder may be used to identify the fish species for the test DNA samples based on the fragment lengths produced in the digestion reactions. Table 7 Expected DNA Fragment Sizes in the Positive Control Restriction Enzyme Expected Product Size (bp) DdeI 117, 332, 340 HaeIII 40, 105, 333 NlaIII 459 The instructions provided here use the default analysis settings in RFLP Matcher. Refer to the software s help system for detailed information on operating the software and interpreting the display.
<ol>
<li>Launch the RFLP Decoder program.</li>
<li>Click File &gt; Open &gt; XAD File. 
The Open dialog box will open.</li>
<li>Select the XAD file for the DNA chip that included the restriction digest reactions. Click Open. 
A dialog box will open listing the samples that were loaded on the chip.</li>
<li>Select three digestion reactions corresponding to one DNA sample and specify the appropriate restriction enzyme for each sample using the drop-down menus under the Enzyme column. In the figure below, the Enzyme column has been filled out for DNA Sample 1. 
<img src="/files/ftp_upload/1871/1871fig4.jpg" alt="DNA restriction analysis chart; enzyme cuts: DdeI, HaeIII, NlaIII; fragment sizes in base pairs." data-altupdatedat="1788235205000" data-alt="figure 4"> 
Figure 4. Specifying Restriction Enzyme for each Sample. </li>
<li>In the field labeled min peak height as % of lower marker", the default value is 10.0%. If needed, this value may be lowered to identify small peaks that was missed, or raised to discard peaks resulting from non-specific noise in the electropherogram. Click Reintegrate after making any adjustments to the min peak height value. 
<img src="/files/ftp_upload/1871/1871fig5.jpg" alt="Chromatography data integration setting, peak reintegration control, minimum peak height adjustment." data-altupdatedat="1788235205000" data-alt="figure 5"> 
Figure 5. Assigning Minimum Peak Height. </li>
<li>Click Calc at the bottom of the dialog box. 
The fragment length data obtained from the Bioanalyzer run will populate the software fields.</li>
<li>In the score drop-down list in the top left corner of the screen, select the appropriate algorithm for analysis of the data. If the fish sample being analyzed consists of a single fish species, select Dice (Nei Li) in the score drop-down list. If the samples may consist of a mixture of species, select Mixture. 
The table at the bottom of the screen labeled combined score lists the best species matches based on the results of all three digestion reactions. The species name as well as the common name are listed in the table (see below). Perfect matches (those with a score of 1) are highlighted in green. Matches highlighted in yellow are considered near matches. You may double-click on a species name to bring up the FishBase webpage for that species. 
<img src="/files/ftp_upload/1871/1871fig6.jpg" alt="Fish species score table, data analysis results, highlighting Thunnus alalunga and related species." data-altupdatedat="1788235205000" data-alt="figure 6"> 
Figure 6. Species Identification Based on Digestion. </li>
<li>In the lower cutoff and match tolerance fields at the top of the screen, you may adjust the default settings to improve the score of the best species match. The lower cutoff value is used to discard any fragments shorter than the length designated in the field. The match tolerance value determines how close in length a fragment must be to the predicted fragment to be considered a match.</li>
<li>Repeat steps 4 through 8 for the remaining DNA samples that were included on this same chip.</li></ol>
8: Representative Results:
An image of a Bioanlyzer gel with restriction digest samples from 4 different fish species is shown in the figure below. The expected results for the positive DNA sample are summarized in the table below. 
<img src="/files/ftp_upload/1871/1871fig7.jpg" alt="gel electrophoresis DNA fragment analysis, fish species comparison, enzyme digestion results diagram" data-altupdatedat="1788235205000" data-alt="figure 7"> 
Figure 7. Bioanalyzer gel image of restriction digest reaction samples. Each gel lane is labeled with the restriction enzyme used in that reaction. 
Expected DNA Fragment Sizes in the Salmon Positive Control
<img src="/files/ftp_upload/1871/1871fig8.jpg" alt="Restriction enzymes table; lists DdeI, HaeIII, NlaIII with expected DNA product sizes (bp)." data-altupdatedat="1788235205000" data-alt="figure 8"> 
Figure 8. Expected DNA Fragment Sizes in the Salmon Positive Control.

<ol><li>Dooley, J. J., Sage, H. D., Brown, H. M., Garrett, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+fish+species+identification+by+use+of+lab-on-a-chip+technology%5BTitle%5D)+AND+%22Food+Control%22%5BJournal%5D)">Improved fish species identification by use of lab-on-a-chip technology.</a> Food Control. 16 (7), 601-607 (2004).</li>
<li>Dooley, J. J., Sage, H. D., Clarke, M. -A. L., Brown, H. M., Garrett, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fish+species+Identification+Using+PCR-RFLP+Analysis+and+Lab-on-a-Chip+Capillary+Electrophoresis%3A+Application+to+Detect+White+Fish+Species+in+Food+Products+and+an+Interlaboratory+Study%5BTitle%5D)+AND+%22J.+Agric.+Food+Chem%22%5BJournal%5D)">Fish species Identification Using PCR-RFLP Analysis and Lab-on-a-Chip Capillary Electrophoresis: Application to Detect White Fish Species in Food Products and an Interlaboratory Study.</a> J. Agric. Food Chem. 53, 3348-3357 (2005).</li>
<li>Dooley, J., Garrett, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+PCR-RFLP+Profiles+for+Fish+Species+Using+the+Agilent+2100+Bioanalyzer%5BTitle%5D)+AND+%22Agilent+Technologies+Application+Note%22%5BJournal%5D)">Determination of PCR-RFLP Profiles for Fish Species Using the Agilent 2100 Bioanalyzer.</a> Agilent Technologies Application Note. , (2005).</li>
<li>Dooley, J. J., Clarke, M. L., Sage, H. D., Brown, H. M., Garrett, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+a+chip-based+capillary+electrophoresis+system+to+enable+simple+PCR-RFLP+identification+of+fish+species%5BTitle%5D)+AND+%22FSA+Final+Report+Q01069%22%5BJournal%5D)">Application of a chip-based capillary electrophoresis system to enable simple PCR-RFLP identification of fish species.</a> FSA Final Report Q01069. , (2004).</li>
</ol>

The authors are employed by Agilent Technologies that produces the reagents and instruments used in this article.

We demonstrate a simple, fast, and accurate DNA-based screening method for the identification of fish species in seafood products. This powerful method provides reproducible, precise results in less than one working day and it can be implemented in commercial testing facilities due to streamlined protocols and simple setup. It is characterized by the generation of objective data that is analyzed using RFLP Decoder software with an expandable database of experimentally-derived fish species profiles.
With routine implementation, this testing method has the potential to prevent mislabeling of seafood products and assist in maintaining accurate records suitable for compliance with governmental regulations.

<table><tbody><tr><td>Agilent 2100 Bioanalyzer (with chip priming station and IKA vortex mixer)</td><td>Agilent Technologies</td><td>G2939AA</td><td></td></tr><tr><td>Agilent 2100 Bioanalyzer laptop &amp;amp; Expert Software</td><td>Agilent Technologies</td><td>G2953CA</td><td>G2950CA - desktop PC &amp;amp; Expert Software</td></tr><tr><td>Agilent Bioanalyzer DNA 1000 Reagent Kit (part #5067-1504)</td><td>Agilent Technologies</td><td>5067-1504</td><td></td></tr><tr><td>Thermal Cycler</td><td>Various</td><td>Various</td><td></td></tr><tr><td>PCR Strip Tubes</td><td>Agilent Technologies</td><td>401428</td><td></td></tr><tr><td>PCR Strip Caps</td><td>Agilent Technologies</td><td>401425</td><td></td></tr><tr><td>96-Well Working Rack</td><td>Agilent Technologies</td><td>410094</td><td></td></tr><tr><td>Fish Species Identification System DNA Isolation Kit</td><td>Agilent Technologies</td><td>Contact</td><td></td></tr><tr><td>Fish Species Identification System PCR-RFLP Reagent Kit</td><td>Agilent Technologies</td><td>Contact</td><td></td></tr><tr><td>RFLP Matcher Software</td><td>Agilent Technologies</td><td>Contact</td><td></td></tr><tr><td>100% ethanol, 200 proof (USP grade or equivalent)</td><td>Various</td><td>Various</td><td></td></tr><tr><td>Sterile, nuclease-free water</td><td>Various</td><td>Various</td><td></td></tr><tr><td>Pipettors</td><td>Various</td><td>Various</td><td></td></tr><tr><td>Incubator or water bath set to 65&amp;#x00B0;C</td><td>Various</td><td>Various</td><td></td></tr><tr><td>Sterile glass bottle or polypropylene tube (e.g. 14-ml BD Falcon polypropylene)</td><td>Various</td><td>Various</td><td></td></tr><tr><td>round-bottom tubes or 50-ml BD Falcon polypropylene conical tubes)</td><td>Various</td><td>Various</td><td></td></tr><tr><td>Vortex mixer</td><td>Various</td><td>Various</td><td></td></tr><tr><td>Microcentrifuge</td><td>Various</td><td>Various</td><td></td></tr><tr><td>Microcentrifuge tubes 1.5ml</td><td>Various</td><td>Various</td><td></td></tr><tr><td>Tube rack 1.5ml</td><td>Various</td><td>Various</td><td></td></tr><tr><td>Pipette Tips</td><td>Various</td><td>Various</td><td></td></tr></tbody></table>

dna-based fish species identification protocol

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. This versatile method employs PCR amplification of genomic DNA extracted from fish samples, followed by restriction fragment length polymorphism (RFLP) analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matched to the correct species using RFLP pattern matching software.
The fish identification method uses a simple, reliable, spin column- based protocol to isolate DNA from fish samples. The samples are treated with proteinase K to release the nucleic acids into solution. DNA is then isolated by suspending the sample in binding buffer and loading onto a micro- spin cup containing a silica- based fiber matrix. The nucleic acids in the sample bind to the fiber matrix. The immobilized nucleic acids are washed to remove contaminants, and total DNA is recovered in a final volume of 100 &mu;l. The isolated DNA is ready for PCR amplification with the provided primers that bind to sequences found in all fish genomes. The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 Bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish from which the DNA sample was prepared, using the RFLP pattern matching software containing a database of experimentally- derived RFLP patterns from commercially relevant fish species.

This publication describes how to use the Agilent Fish Species Identification System to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis.

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

The Fish Species Identification System is a simple, fast, and accurate DNA based method to identify the fish species present in a given sample. Fish samples are treated with proteinase K to release nucleic acids into solution. The fish genomic DNA is then isolated and PCR amplified using primers that bind to sequences found in all fish genomes.The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish using restriction fragment length polymorphism, or RFLP pattern matching software. Hi, I'm Rachel Formosa from Agilent Technologies.Today we will show you a procedure for DNA-based fish species identification. This procedure is a screening method that allows you to identify the fish species present in either fresh, frozen, minced, cooked, dried or otherwise processed samples. And this can be accomplished in less than one workday.We use this procedure to study seafood authenticity, which is very important to the seafood industry as substitution and mislabeling can have significant economic, environmental and food safety consequences. Plus, it can be especially difficult to determine fish species by visual identification and other traditional methods. Once a fish has been processed, DNA testing provides a much more accurate and reliable result.So let's get started. Begin preparing samples for DNA extraction by placing 10 to 1000 milligrams of each raw or cooked fish tissue sample in 1.5 milliliter micro centrifuge tubes to each fish. Sample at 220 microliters of freshly prepared proteinase case solution and pipette up and down.Incubate the tubes at 65 degrees Celsius for 10 minutes following the incubation centrifuge for three to five minutes at 14, 000 Gs to pellet any undigested tissues. Next carefully transfer 150 microliters of each supernatant to a fresh 1.5 milliliter tube, avoiding any undigested material at the bottom and oily material present at the top. The supernatant will now be used for genomic DNA extraction.Begin the genomic DNA extraction by adding 500 microliters of the nucleic acid binding buffer to each sample. This will bring the total sample volume to 650 microliters. Vortex the sample until homogenized.Next, transfer each sample to a DNA binding spin cup that has been seated in one of the two milliliter receptacle tubes provided in the kit. Snap the cap of the tube onto the top of the spin cup. Spin the samples for one minute at 14, 000 Gs to load the DNA onto the spin cup matrix.After the centrifugation, remove the spin cups, discard the filtrate, and place the cups back in the receptacle tubes. Add 500 microliters of one x high salt wash buffer. Cap the tubes and centrifuge again after the centrifugation.Discard the filtrate and again, place the spin cups back in the receptacle tubes. Now add 500 microliters of 80%ethanol. Cap the tube and spin at 14, 000 Gs for one minute.Repeat the ethanol wash two more times after the third wash in 80%ethanol again, discard the filtrates. Replace the spin cups in the receptacle tubes and this time spin for two minutes to dry the fiber matrix. Now transfer the spin cups to 1.5 milliliter collection tubes.Add 100 microliters evolution buffer to each spin cup directly on the fiber matrix inside the cup. Then snap the collection tube caps onto the spin cups and incubate them at room temperature for one minute. Following the incubation, spin the samples at maximum speed for one minute.Next, discard the spin cup and cap the tubes. If DNA concentrations are measured concentrations ranging from five nanograms per microliter to 500 nanograms per microliter are expected. The DNA may now be stored at four degrees Celsius for up to one month for long-term storage, place the DNA at minus 20 or minus 80 degrees Celsius.Perform PCR using the primers and reagents provided in the P-C-R-R-F-L-P reagent kit. According to the accompanying written protocol, it is recommended to run each sample, including the positive anti no template control in duplicate while the PCR is running. Prepare restriction enzyme master mixes for the digestions with DTE E one HAE three, and NL three by combining the following components in order, 1.5 microliters of sterile distilled water 0.5 microliters of 10 x enzyme buffer and 0.5 microliters of 10 x enzyme.Prepare enough for all samples plus one reaction volume, excess fourex three reagent mixture. Then distribute 2.5 microliters to each reaction tube labeled with the sample and enzyme name. Once the PCR is complete, remove the samples from the thermal cycler and place them on ice.Add 2.5 microliters of each PCR product to each of the three restriction digest tubes for that reaction. DDE one HAE three, and NLA three. Next vortex.Briefly centrifuge and incubate the digestions at 37 degrees Celsius for two hours. If more convenient, the digestions can also be incubated overnight. Following the digestion, incubate the reactions at 65 degrees Celsius for 15 minutes.Then add one microliter of 60 millimolar EDTA to each tube to terminate the reaction and vortex. Using the Agilent bioanalyzer reagent kit, prepare and load the restriction digest into the wells of A DNA chip according to the kit guide. For each sample, all three digests should be loaded on the same chip.Then vortex the chip and load it into the machine. After the run has finished, go to the assay context and select the chip summary tab. In the sample name field, enter a sample name for all 12 wells of the chip to identify the fish species for the test DNA samples.Launch the RFLP decoder program by clicking file. Then open XAD file. The open dialogue box will open.Now select the XAD file for the DNA chip used. And click open to see a list of the samples loaded on the chip. Select the three digests corresponding to the first DNA sample.Under the enzyme column, specify the restriction enzymes that were used in the field labeled min peak height as percent of lower marker. The default value is 10%if needed. This value may be lowered to identify small peaks that were missed or raised to discard peaks resulting from non-specific noise in the electropherogram.After making any adjustments to the min peak height value, click reintegrate. Now click calc at the bottom of the dialogue box. The fragment length data obtained from the bioanalyzer run will populate the software fields.Next in the score dropdown list in the top left corner of the screen, select dice. If the fish sample consists of a single fish species or mixture, if the sample may consist of a mixture, the table at the bottom of the screen labeled combined score list. The best species matches based on the results of all three digestion reactions.Perfect matches with a score of one are highlighted in green. Near matches are highlighted in yellow at the top of the screen. The lower cutoff value designates the minimum fragment size used in the analysis and the match tolerance value determines how close in length a fragment must be to the predicted fragment to be considered a match.The value shown are the default settings and may be adjusted. Repeat these steps for analysis of the remaining DNA samples on the tip. DNA samples from four different fish species were isolated, amplified, and digested using the method described in this video.A bioanalyzer gel showing the restriction digest samples is shown here using the Agilent Bioanalyzer and RFLP decoder. Software bending patterns are used to identify the fish here. The first sample is correctly identified as trout, the second as tuna, the third as rock soil.And the last sample as specific cod. We've just shown you how to perform DNA based fish species identification using agilent's P-C-R-R-F-L-P method. When doing this procedure, it's important to avoid cross-contamination of samples since the method is so sensitive.So that's it. Now you know how to quickly, easily, and accurately identify the fish species present in your samples. Thanks for watching and good luck with your experiments.

dna-based-fish-species-identification-protocol

Research

JoVE Journal

Biology

This article describes DNA extraction from zebrafish tail clip. The protocol demonstrates a rapid and efficient method to analyze zebrafish genotypes.

DNA Extraction from Tail Clip: A Method Used in Zebrafish Genotyping

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

Robust 3D DNA FISH Using Directly Labeled Probes

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

An improved polymerase chain reaction-restriction fragment length polymorphism method for genotyping pufferfish species by liquid chromatography/mass spectrometry is described. A reverse-phase silica monolith column is employed for separating digested amplicons. This method can elucidate the monoisotopic masses of oligonucleotides, which is useful for identifying base composition.

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Laboratory housing of turquoise killifish can be scaled up to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The health of wild fishes can be used as an indicator of aquatic ecosystem health. Necropsy-based fish health assessments provide documentation of visible lesions or abnormalities, data used to calculate condition indices as well as the opportunity to collect tissues for microscopic evaluation, gene expression and other more in-depth analyses.

Necropsy-based Wild Fish Health Assessment

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Environmental DNA assays require rigorous design, testing, optimization and validation before the collection of field data can begin. Here, we present a protocol to take users through each step of designing a species-specific, probe-based qPCR assay for the detection and quantification of a target species DNA from environmental samples.

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

This work describes a protocol for quantifying craniofacial cartilage shape using free software (tpsDigs2, MorphoJ, and PAST) to measure changes in facial structure in zebrafish larvae.

Morphometric Analyses of Shape: The Analysis Software Toolbox for Craniofacial Shape Quantification in Zebrafish

Learn DNA-based fish species identification using PCR and RFLP analysis with the Agilent system. This method enables accurate seafood authenticity testing for processed samples in less than one workday.

DNA-based Fish Species Identification Protocol

Explore More Videos