DNA-based Fish Species Identification Protocol

The Fish Species Identification System is a simple, fast, and accurate DNA based method to identify the fish species present in a given sample. Fish samples are treated with proteinase K to release nucleic acids into solution. The fish genomic DNA is then isolated and PCR amplified using primers that bind to sequences found in all fish genomes.

The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish using restriction fragment length polymorphism, or RFLP pattern matching software. Hi, I'm Rachel Formosa from Agilent Technologies.

Today we will show you a procedure for DNA-based fish species identification. This procedure is a screening method that allows you to identify the fish species present in either fresh, frozen, minced, cooked, dried or otherwise processed samples. And this can be accomplished in less than one workday.

We use this procedure to study seafood authenticity, which is very important to the seafood industry as substitution and mislabeling can have significant economic, environmental and food safety consequences. Plus, it can be especially difficult to determine fish species by visual identification and other traditional methods. Once a fish has been processed, DNA testing provides a much more accurate and reliable result.

So let's get started. Begin preparing samples for DNA extraction by placing 10 to 1000 milligrams of each raw or cooked fish tissue sample in 1.5 milliliter micro centrifuge tubes to each fish. Sample at 220 microliters of freshly prepared proteinase case solution and pipette up and down.

Incubate the tubes at 65 degrees Celsius for 10 minutes following the incubation centrifuge for three to five minutes at 14, 000 Gs to pellet any undigested tissues. Next carefully transfer 150 microliters of each supernatant to a fresh 1.5 milliliter tube, avoiding any undigested material at the bottom and oily material present at the top. The supernatant will now be used for genomic DNA extraction.

Begin the genomic DNA extraction by adding 500 microliters of the nucleic acid binding buffer to each sample. This will bring the total sample volume to 650 microliters. Vortex the sample until homogenized.

Next, transfer each sample to a DNA binding spin cup that has been seated in one of the two milliliter receptacle tubes provided in the kit. Snap the cap of the tube onto the top of the spin cup. Spin the samples for one minute at 14, 000 Gs to load the DNA onto the spin cup matrix.

After the centrifugation, remove the spin cups, discard the filtrate, and place the cups back in the receptacle tubes. Add 500 microliters of one x high salt wash buffer. Cap the tubes and centrifuge again after the centrifugation.

Discard the filtrate and again, place the spin cups back in the receptacle tubes. Now add 500 microliters of 80%ethanol. Cap the tube and spin at 14, 000 Gs for one minute.

Repeat the ethanol wash two more times after the third wash in 80%ethanol again, discard the filtrates. Replace the spin cups in the receptacle tubes and this time spin for two minutes to dry the fiber matrix. Now transfer the spin cups to 1.5 milliliter collection tubes.

Add 100 microliters evolution buffer to each spin cup directly on the fiber matrix inside the cup. Then snap the collection tube caps onto the spin cups and incubate them at room temperature for one minute. Following the incubation, spin the samples at maximum speed for one minute.

Next, discard the spin cup and cap the tubes. If DNA concentrations are measured concentrations ranging from five nanograms per microliter to 500 nanograms per microliter are expected. The DNA may now be stored at four degrees Celsius for up to one month for long-term storage, place the DNA at minus 20 or minus 80 degrees Celsius.

Perform PCR using the primers and reagents provided in the P-C-R-R-F-L-P reagent kit. According to the accompanying written protocol, it is recommended to run each sample, including the positive anti no template control in duplicate while the PCR is running. Prepare restriction enzyme master mixes for the digestions with DTE E one HAE three, and NL three by combining the following components in order, 1.5 microliters of sterile distilled water 0.5 microliters of 10 x enzyme buffer and 0.5 microliters of 10 x enzyme.

Prepare enough for all samples plus one reaction volume, excess fourex three reagent mixture. Then distribute 2.5 microliters to each reaction tube labeled with the sample and enzyme name. Once the PCR is complete, remove the samples from the thermal cycler and place them on ice.

Add 2.5 microliters of each PCR product to each of the three restriction digest tubes for that reaction. DDE one HAE three, and NLA three. Next vortex.

Briefly centrifuge and incubate the digestions at 37 degrees Celsius for two hours. If more convenient, the digestions can also be incubated overnight. Following the digestion, incubate the reactions at 65 degrees Celsius for 15 minutes.

Then add one microliter of 60 millimolar EDTA to each tube to terminate the reaction and vortex. Using the Agilent bioanalyzer reagent kit, prepare and load the restriction digest into the wells of A DNA chip according to the kit guide. For each sample, all three digests should be loaded on the same chip.

Then vortex the chip and load it into the machine. After the run has finished, go to the assay context and select the chip summary tab. In the sample name field, enter a sample name for all 12 wells of the chip to identify the fish species for the test DNA samples.

Launch the RFLP decoder program by clicking file. Then open XAD file. The open dialogue box will open.

Now select the XAD file for the DNA chip used. And click open to see a list of the samples loaded on the chip. Select the three digests corresponding to the first DNA sample.

Under the enzyme column, specify the restriction enzymes that were used in the field labeled min peak height as percent of lower marker. The default value is 10%if needed. This value may be lowered to identify small peaks that were missed or raised to discard peaks resulting from non-specific noise in the electropherogram.

After making any adjustments to the min peak height value, click reintegrate. Now click calc at the bottom of the dialogue box. The fragment length data obtained from the bioanalyzer run will populate the software fields.

Next in the score dropdown list in the top left corner of the screen, select dice. If the fish sample consists of a single fish species or mixture, if the sample may consist of a mixture, the table at the bottom of the screen labeled combined score list. The best species matches based on the results of all three digestion reactions.

Perfect matches with a score of one are highlighted in green. Near matches are highlighted in yellow at the top of the screen. The lower cutoff value designates the minimum fragment size used in the analysis and the match tolerance value determines how close in length a fragment must be to the predicted fragment to be considered a match.

The value shown are the default settings and may be adjusted. Repeat these steps for analysis of the remaining DNA samples on the tip. DNA samples from four different fish species were isolated, amplified, and digested using the method described in this video.

A bioanalyzer gel showing the restriction digest samples is shown here using the Agilent Bioanalyzer and RFLP decoder. Software bending patterns are used to identify the fish here. The first sample is correctly identified as trout, the second as tuna, the third as rock soil.

And the last sample as specific cod. We've just shown you how to perform DNA based fish species identification using agilent's P-C-R-R-F-L-P method. When doing this procedure, it's important to avoid cross-contamination of samples since the method is so sensitive.

So that's it. Now you know how to quickly, easily, and accurately identify the fish species present in your samples. Thanks for watching and good luck with your experiments.