1、Journal of Visualized ECopyright 2010 Journal of Visualized Experiments and Agilent Technologies,Inc.April 2010|38|e1871|Page 1 of 7Video ArticleDNA-based Fish Species Identification ProtocolRachel Formosa1,Harini Ravi1,Scott Happe1,Danielle Huffman1,Natalia Novoradovskaya1,Robert Kincaid1,Steve Gar
2、rett11Agilent TechnologiesCorrespondence to:Rachel Formosa at URL:http:/ Biology,Issue 38,seafood,fish,mislabeling,authenticity,PCR,Bioanalyzer,food,RFLP,identityDate Published:4/28/2010Citation:Formosa,R.,Ravi,H.,Happe,S.,Huffman,D.,Novoradovskaya,N.,Kincaid,R.,Garrett,S.DNA-based Fish Species Iden
3、tificationProtocol.J.Vis.Exp.(38),e1871,doi:10.3791/1871(2010).AbstractWe have developed a fast,simple,and accurate DNA-based screening method to identify the fish species present in fresh and processedseafood samples.This versatile method employs PCR amplification of genomic DNA extracted from fish
4、 samples,followed by restrictionfragment length polymorphism(RFLP)analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matchedto the correct species using RFLP pattern matching software.The fish identification method uses a simple,reliable,spin column-based
5、 protocol to isolate DNA from fish samples.The samples are treatedwith proteinase K to release the nucleic acids into solution.DNA is then isolated by suspending the sample in binding buffer and loading onto amicro-spin cup containing a silica-based fiber matrix.The nucleic acids in the sample bind
6、to the fiber matrix.The immobilized nucleic acids arewashed to remove contaminants,and total DNA is recovered in a final volume of 100 l.The isolated DNA is ready for PCR amplification with theprovided primers that bind to sequences found in all fish genomes.The PCR products are then digested with t
7、hree different restriction enzymesand resolved on the Agilent 2100 Bioanalyzer.The fragment lengths produced in the digestion reactions can be used to determine the speciesof fish from which the DNA sample was prepared,using the RFLP pattern matching software containing a database of experimentally-
8、derivedRFLP patterns from commercially relevant fish species.Video LinkThe video component of this article can be found at http:/ the Fish Samples for DNA Extraction1.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
9、microcentrifuge tube.Use the figure below as a guideline for estimating the weight of a raw fish sample based on sample size.Figure 1.Mass Estimations of Fish Samples Based on Sample Size.2.Pre-warm the Proteinase K Digestion Buffer to 65C for 5 minutes in an incubator or water bath.3.Prepare a work
10、ing solution of Proteinase K by combining 200 l of Proteinase K Digestion Buffer and 20 l of Proteinase K per sample.Note:Prepare a fresh working solution of Proteinase K before each use.4.Add 220 l of the Proteinase K working solution to each 1.5-ml tube of fish sample.Incubate the tubes at 65C for
11、 10 minutes in an incubatoror water bath.5.Spin the tubes in a microcentrifuge for 3 5 minutes at 14,000 x g to pellet any undigested tissues.Journal of Visualized ECopyright 2010 Journal of Visualized Experiments and Agilent Technologies,Inc.April 2010|38|e1871|Page 2 of 76.Transfer 150 l of each s
12、upernatant into a fresh 1.5-ml tube.Avoid transferring any undigested material from the bottom of the tube orany oily material that may be present at the top of the tube.These tubes of supernatant are the samples from which genomic DNA will beextracted.2:Extract Genomic DNA1.In a sterile container(p
13、olypropylene tube or glass bottle),prepare a working solution of sulfolane and Nucleic Acid Binding Buffer bycombining 320 l of 90%sulfolane and 170 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 exa
14、mine 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.2.Add 490 l of the sulfolane/Binding Buffer mixture(prepared in step 1 above)to each fish sample.Vortex or pipet the sample repeatedly untilhomogenized.The
15、 addition of this mixture will bring the total volume of each sample to 640 l.3.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 tubeonto the top of the spin cup.4.Spin the samples in a microcentrifuge for 1 minute at
16、14,000 x g to load the DNA onto the spin cup matrix.5.Remove and retain the spin cups and discard the filtrates.For each sample,replace the spin cup in the receptacle tube,then add 500 l of1x High Salt Wash Buffer and cap the tube.6.Spin the samples in a microcentrifuge at 14,000 x g for 1 minute.7.
17、Remove and retain the spin cups and discard the filtrates.For each sample,replace the spin cup in the receptacle tube,then add 500 l of80%ethanol and cap the tube.8.Spin the samples in a microcentrifuge at 14,000 x g for 1 minute.9.Repeat steps 7 and 8 two more times for a total of 3 washes with 500
18、 l of 80%ethanol.10.After the third wash in 80%ethanol,remove and retain the spin cups and discard the filtrates.Replace the spin cups in their receptacletubes and spin in a microcentrifuge for 2 minutes at 14,000 x g to dry the fiber matrix.11.Transfer the spin cups to 1.5-m1 collection tubes.Add 1
19、00 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.12.Spin the samples in a microcentrifuge at maximum speed for 1 minute.13.The purified DNA is in the Elution Buff
20、er in the microcentrifuge tube.Discard the spin cup and cap the tubes.The DNA may be stored at 4Cfor up to one month.For long-term storage,store the DNA at 20C or 80C.14.If desired,you may measure the concentration of the DNA samples in a spectrophotometer.The genomic DNA extraction protocol typical
21、ly yields samples with a concentration ranging from 5 ng/l to 500 ng/l.The PCR-RFLP protocolworks wells with DNA samples ranging anywhere from 0.05 ng/l to 2000 ng/l.3:Set up the PCR Reactions1.Prepare a 50 ng/l dilution of the positive control salmon DNA by combining 8 l of the DNA stock with 32 l
22、of sterile,DNase-free water.Vortex to mix.The diluted sample may be stored at 4C for future use.2.Prepare the reactions by combining the components in the table below in order.Prepare a single reagent mixture for all PCR reactions thatwill be run simultaneously by scaling up the volumes listed in th
23、e table.In addition to the test DNA samples,include a positive control reactionand a no-template control reaction.Preparing duplicate PCR reactions for each test DNA sample is recommended.Prepare enough reagentmixture for all your reactions plus one reaction volume excess.For example,if you have 5 t
24、est DNA samples,prepare enough reagent mixture for either 8 reactions(5 test reactions,1 positive control,1no-template control,and 1 excess)or 13 reactions if you are including duplicates of the test reactions(10 duplicate test reactions,1 positivecontrol,1 no-template control and 1 excess).PCR Reag
25、ent MixtureComponentVolume 1 ReactionVolume 5 ReactionsdH2O,sterile9 l45 l2x PCR Master Mix12.5 l62.5 lPrimer Mix2.5 l12.5 lTotal volume24 l120 l3.Vortex the reagent mixture well,then distribute 24 l to each individual thin-walled PCR reaction tube.4.Add 1 l of the diluted positive control DNA to th
26、e positive control reaction tube.To the test sample tubes,add 1 l of test DNA sample.Forthe no-template control reaction,add 1 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 pipettin
27、g thecontents of the tube up and down.5.Cap the reaction tubes,vortex the tubes to mix and centrifuge the tubes briefly.Journal of Visualized ECopyright 2010 Journal of Visualized Experiments and Agilent Technologies,Inc.April 2010|38|e1871|Page 3 of 74:Run the PCR Protocol1.Place the reactions in t
28、he thermal cycler and run the PCR program shown below.PCR Cycling ProtocolSegmentNumber of CyclesTemperatureDuration1195C5 minutes24095C 50C 72C30 seconds 30 seconds 30 seconds3172C7 minutes5:Digest PCR Products with Restriction Enzymes1.Label the 0.5-ml tubes or 0.2-ml strip tubes that are to be us
29、ed for the restriction digest reactions.Each PCR reaction will be digested withthree different restriction enzymes:DdeI,HaeIII and NlaIII.Therefore,for each PCR reaction,label three separate tubes with the name of thePCR sample and the name of the restriction enzyme.2.Prepare the reagent mixture for
30、 the DdeI digestions by combining the components below in order.Prepare a single reagent mixture for allDdeI digestion reactions(plus at least one reaction volume excess)using multiples of each component.Once the PCR is complete,the PCRreactions are treated with restriction enzymes for restriction f
31、ragment length polymorphism(RFLP)analysis.DdeI Digestion Reagent MixtureComponentVolumedH2O,sterile1.5 l10 x DdeI Buffer0.5 l10 x DdeI enzyme0.5 l3.Vortex the reagent mixture well,then distribute 2.5 l to the individual reaction tubes that were labeled for DdeI.4.Prepare the reagent mixture for the
32、HaeIII digestions by combining the components below in order.Prepare a single reagent mixture for allHaeIII digestion reactions(plus at least one reaction volume excess)using multiples of each component.HaeIII Digestion Reagent MixtureComponentVolumedH2O,sterile1.5 l10 x HaeIII Buffer0.5 l10 x HaeII
33、I enzyme0.5 l5.Vortex the reagent mixture well,then distribute 2.5 l to the individual reaction tubes that were labeled for HaeIII.6.Prepare the reagent mixture for the NlaIII digestions by combining the components below in order.Prepare a single reagent mixture for allNlaIII digestion reactions(plu
34、s at least one reaction volume excess)using multiples of each component.NlaIII Digestion Reagent MixtureComponentVolumedH2O,sterile1.5 l10 x NlaIII Buffer0.5 l10 x NlaIII enzyme0.5 l7.Vortex the reagent mixture well,then distribute 2.5 l to the individual reaction tubes that were labeled for NlaIII.
35、8.For each digestion reaction,add 2.5 l of the appropriate PCR product to the labeled tubes.All of the test PCR reactions as well as thepositive control reaction need to be digested with all three restriction enzymes.9.Vortex the digestion reactions and then briefly centrifuge the tubes.10.Incubate
36、all the digestion reactions at 37C for 2 hours.This incubation can be performed in the thermal cycler.If desired,reactions may beleft at 37C overnight.11.Transfer the reactions to 65C for 15 minutes.This step can be performed in the thermal cycler.12.Add 1 l of 60 mM EDTA(provided with the kit)to ea
37、ch reaction and vortex well.Journal of Visualized ECopyright 2010 Journal of Visualized Experiments and Agilent Technologies,Inc.April 2010|38|e1871|Page 4 of 76:Analyze the restriction digest patterns1.Prepare the gel-dye mix,place a DNA chip on the chip priming station,and load the mix onto the DN
38、A chip.2.Pipet 5 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.3.Pipet 1 l of DNA ladder into the well marked with a ladder symbol.DNA ladder is provided in the DN
39、A 1000 Reagent Kit in a yellow-capped tube.4.For each of the DNA samples,pipet 1 l of each restriction digest reaction into one of the 12 sample wells on the chip according to theguidelines in the figure below.Using this approach,wells 1-3 are for the 3 digestion reactions for the positive control s
40、ample,wells 4-6 arefor 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 3digestion reactions for test sample 3.Analyze the restriction digest reactions on the Agilent 2100 Bioanalyzer to determine the fragmentlength
41、s produced during digestion.The Agilent DNA 1000 Kit Guide has complete instructions for running the lab chips on the Bioanalyzer.Abrief protocol is provided below for your convenience.Figure 2.Organization of Samples on Chip.If you have less than 4 DNA samples,pipet 1 l of water into the unused wel
42、ls.If you have more than 4 DNA samples,you will need to runmore than one chip.Note that it is not necessary to run the positive control sample on every chip.5.Place the chip horizontally in the adapter of the IKA vortex mixer and vortex for 60 seconds at 2400 rpm.6.Insert the chip into the Agilent 2
43、100 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 hasfailed to detect a genuine peak,you may lower the Heig
44、ht Threshold setpoint to a value that allows the algorithm to identify the peak.7.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 asshown in the figure below.Figure 3.Sample Name Entry in Chip Summary Tab.Journal of Vi
45、sualized ECopyright 2010 Journal of Visualized Experiments and Agilent Technologies,Inc.April 2010|38|e1871|Page 5 of 77:Identify the test sample species using RFLP MatcherThe Agilent software application RFLP Decoder may be used to identify the fish species for the test DNA samples based on the fra
46、gment lengthsproduced in the digestion reactions.Table 7 Expected DNA Fragment Sizes in the Positive Control Restriction Enzyme Expected ProductSize(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
47、 s help system for detailed information on operating the software and interpreting the display.1.Launch the RFLP Decoder program.2.Click File Open XAD File.The Open dialog box will open.3.Select the XAD file for the DNA chip that included the restriction digest reactions.Click Open.A dialog box will
48、 open listing the samples that were loaded on the chip.4.Select three digestion reactions corresponding to one DNA sample and specify the appropriate restriction enzyme for each sample using thedrop-down menus under the Enzyme column.In the figure below,the Enzyme column has been filled out for DNA
49、Sample 1.Figure 4.Specifying Restriction Enzyme for each Sample.5.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 smallpeaks that was missed,or raised to discard peaks resulting from non-specific noise in the electrop
50、herogram.Click Reintegrate after makingany adjustments to the min peak height value.Figure 5.Assigning Minimum Peak Height.6.Click Calc at the bottom of the dialog box.The fragment length data obtained from the Bioanalyzer run will populate the software fields.7.In the score drop-down list in the to
