Name: dna-based fish species identification protocol
Uploaded: 2010-04-28T17:00:00
Duration: 9 min 15 s
Description: Watch this Scientific Journal Video about DNA-based Fish Species Identification Protocol at JoVE.com

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.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Agilent 2100 Bioanalyzer (with chip priming station and IKA vortex mixer)</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>G2939AA</td>
<td>&nbsp;</td>
<tr>
<td>Agilent 2100 Bioanalyzer laptop &amp; Expert Software</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>G2953CA</td>
<td>G2950CA - desktop PC &amp; Expert Software</td>
</tr>
<tr>
<td>Agilent Bioanalyzer DNA 1000 Reagent Kit (part #5067-1504)</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>5067-1504</td>
<td>&nbsp;</td>
<tr>
<td>Thermal Cycler</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>PCR Strip Tubes</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>401428</td>
<td>&nbsp;</td>
<tr>
<td>PCR Strip Caps</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>401425</td>
<td>&nbsp;</td>
<tr>
<td>96-Well Working Rack</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>410094</td>
<td>&nbsp;</td>
<tr>
<td>Fish Species Identification System DNA Isolation Kit</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>Contact</td>
<td>&nbsp;</td>
<tr>
<td>Fish Species Identification System PCR-RFLP Reagent Kit</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>Contact</td>
<td>&nbsp;</td>
<tr>
<td>RFLP Matcher Software</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>Contact</td>
<td>&nbsp;</td>
<tr>
<td>100% ethanol, 200 proof (USP grade or equivalent)</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Sterile, nuclease-free water</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Pipettors</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Incubator or water bath set to 65&deg;C</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Sterile glass bottle or polypropylene tube (e.g. 14-ml BD Falcon polypropylene)</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>round-bottom tubes or 50-ml BD Falcon polypropylene conical tubes)</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Vortex mixer</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Microcentrifuge</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Microcentrifuge tubes 1.5ml</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Tube rack 1.5ml</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Pipette Tips</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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

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Biology

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

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

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels (Bivalvia: Unionida)

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater mussels are among the most imperiled organisms in the world, but information about early larval (glochidial) development and brooding periods is still lacking for many species. Previous studies have focused on the complex life history stage when female mussels are ready to parasitize host fish, but few studies have focused on the brooding period and timing of larval development. The protocol described here allows researchers to non-lethally evaluate the state of gravidity for female mussels. The results of this study show that this method does not affect a female mussel&rsquo;s ability to stay gravid or become gravid again after sampling has been performed. The advantage of this method may permit its use on federally threatened or endangered species or other populations of high conservation concern. This protocol can be adapted for use on both preserved or live individuals and was tested on a variety of mussel species. The database provided is a repository for a breadth of information on timing of reproductive habits and will facilitate future freshwater mussel research, conservation, and recovery efforts.

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels Bivalvia: Unionida

Freshwater mussel conservation is dependent on monitoring reproductive patterns and processes of species. This study standardizes a non-lethal protocol for sampling gill contents, characterizing larval development, and providing a digital repository for data collected. This protocol-database package will be an important tool for mussel researchers in recovery of imperiled species.

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater ...

Assessing Agrochemical Risk to Mated Honey Bee Queens

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not accurately capture the effects of agrochemical exposure on honey bee queens. As the sole producer of fertilized eggs inside a honeybee colony, the queen is arguably the most important single member of a functioning colony unit. Therefore, understanding how agrochemicals affect queen health and productivity should be considered a critical aspect of pesticide risk assessment. Here, an adapted method is presented to expose honey bee queens and worker queen attendants to agrochemical stressors administered through a worker diet, followed by tracking egg production in the laboratory and assessing first instar eclosion using a specialized cage, referred to as a Queen Monitoring Cage. To illustrate the method&#39;s intended use, results of an experiment in which worker queen attendants were fed diet containing sublethal doses of imidacloprid and effects on queens were monitored are described.

This protocol was developed to enhance the understanding of how agrochemicals affect honey bee (Apis mellifera) reproduction by establishing methods to expose honey bee queens and their worker caretakers to agrochemicals in a controlled, laboratory setting and carefully monitoring their relevant responses.

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not ...