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="figure 1" /> 
Figure 1. Mass Estimations of Fish Samples Based on Sample Size. </li>
<li>Pre-warm the Proteinase K Digestion Buffer to 65&deg;C for 5 minutes in an incubator or water bath.</li>
<li>Prepare a working solution of Proteinase K by combining 200 &mu;l of Proteinase K Digestion Buffer and 20 &mu;l of Proteinase K per sample. 
Note: Prepare a fresh working solution of Proteinase K before each use.</li>
<li>Add 220 &mu;l of the Proteinase K working solution to each 1.5-ml tube of fish sample. Incubate the tubes at 65&deg;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 &mu;l 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 &mu;l of 90% sulfolane and 170 &mu;l 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 &mu;l 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 &mu;l.</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 &mu;l 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 &mu;l 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 &mu;l 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 &mu;l 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&deg;C for up to one month. For long-term storage, store the DNA at 20&deg;C or 80&deg;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/&mu;l to 500 ng/&mu;l. The PCR-RFLP protocol works wells with DNA samples ranging anywhere from 0.05 ng/&mu;l to 2000 ng/&mu;l.
3: Set up the PCR Reactions
<ol> 
<li>	Prepare a 50 ng/&mu;l dilution of the positive control salmon DNA by combining 8 &mu;l of the DNA stock with 32 &mu;l of sterile, DNase-free water. Vortex to mix. 
The diluted sample may be stored at 4&deg;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 &mu;l</td>
 <td>45 &mu;l</td>
 </tr>
 <tr>
 <td>2x PCR Master Mix</td>
 <td>12.5 &mu;l</td>
 <td>62.5 &mu;l</td>
 </tr>
 <tr>
 <td>Primer Mix</td>
 <td>2.5 &mu;l</td>
 <td>12.5 &mu;l</td>
 </tr>
 <tr>
 <td>Total volume</td>
 <td>24 &mu;l</td>
 <td>120 &mu;l</td>
 </tr>
 </tbody>
</table>
</li>
<li>Vortex the reagent mixture well, then distribute 24 &mu;l to each individual thin-walled PCR reaction tube.</li>
<li>Add 1 &mu;l of the diluted positive control DNA to the positive control reaction tube. To the test sample tubes, add 1 &mu;l of test DNA sample. For the no-template control reaction, add 1 &mu;l 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&deg;C</td>
 <td>5 minutes</td>
 </tr>
 <tr>
 <td>2</td>
 <td>40</td>
 <td>95&deg;C 50&deg;C 72&deg;C</td>
 <td>30 seconds 30 seconds 30 seconds</td>
 </tr>
 <tr>
 <td>3</td>
 <td>1</td>
 <td>72&deg;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 &mu;l</td>
 </tr>
 <tr>
 <td>10x DdeI Buffer</td>
 <td>0.5 &mu;l</td>
 </tr>
 <tr>
 <td>10x DdeI enzyme</td>
 <td>0.5 &mu;l</td>
 </tr>
 </tbody>
</table>
</li>
<li>Vortex the reagent mixture well, then distribute 2.5 &mu;l 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 &mu;l</td>
 </tr>
 <tr>
 <td>10x HaeIII Buffer</td>
 <td>0.5 &mu;l</td>
 </tr>
 <tr>
 <td>10x HaeIII enzyme</td>
 <td>0.5 &mu;l</td>
 </tr>
 </tbody>
</table>
</li>
<li>Vortex the reagent mixture well, then distribute 2.5 &mu;l 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 &mu;l</td>
 </tr>
 <tr>
 <td>10x NlaIII Buffer</td>
 <td>0.5 &mu;l</td>
 </tr>
 <tr>
 <td>10x NlaIII enzyme</td>
 <td>0.5 &mu;l</td>
 </tr>
 </tbody>
</table>
</li>
<li>Vortex the reagent mixture well, then distribute 2.5 &mu;l to the individual reaction tubes that were labeled for NlaIII.</li>
<li>For each digestion reaction, add 2.5 &mu;l 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&deg;C for 2 hours. This incubation can be performed in the thermal cycler. If desired, reactions may be left at 37&deg;C overnight.</li>
<li>Transfer the reactions to 65&deg;C for 15 minutes. This step can be performed in the thermal cycler.</li>
<li>Add 1 &mu;l 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 &mu;l 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 &mu;l 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 &mu;l 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="figure 2" /> 
Figure 2. Organization of Samples on Chip. 
If you have less than 4 DNA samples, pipet 1 &mu;l 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="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="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="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="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="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="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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+fish+species+identification+by+use+of+lab-on-a-chip+technology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+PCR-RFLP+Profiles+for+Fish+Species+Using+the+Agilent+2100+Bioanalyzer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+chip-based+capillary+electrophoresis+system+to+enable+simple+PCR-RFLP+identification+of+fish+species.">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

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.

Watch this Scientific Journal Video about DNA-based Fish Species Identification Protocol at JoVE.com

DNA-based Fish Species Identification Protocol

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. ...

dna-based-fish-species-identification-protocol

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

29.3K Views.  Agilent Technologies. 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.

Video: DNA-based Fish Species Identification Protocol

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

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 &#177; 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.

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.

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

Genetics

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

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

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

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.

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

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

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The use of environmental DNA (eDNA) samples for detecting macrobiota is one such group of methods that is rapidly becoming popular and being implemented in national management programs. Here we focus on the development of species-specific targeted assays for probe-based quantitative PCR (qPCR) applications. Using probe-based qPCR offers greater specificity than is possible with primers alone. Furthermore, the ability to quantify the amount of DNA in a sample can be useful in our understanding of the ecology of eDNA and the interpretation of eDNA detection patterns in the field. Careful consideration is needed in the development and testing of these assays to ensure the sensitivity and specificity of detecting the target species from an environmental sample. In this protocol we will delineate the steps needed to design and test probe-based assays for the detection of a target species; including creation of sequence databases, assay design, assay selection and optimization, testing assay performance, and field validation. Following these steps will help achieve an efficient, sensitive, and specific assay that can be used with confidence. We demonstrate this process with our assay designed for populations of the mucket (Actinonaias ligamentina), a freshwater mussel species found in the Clinch River, USA.

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.

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The ...

Environment

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Developmental Biology

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

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

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

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

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

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

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.

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

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

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;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.

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...

DNA-based Fish Species Identification Protocol

Summary

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Chapters in this video

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

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

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

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

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

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

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

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

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing