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Page 1: Lab Investigation 3: BLAST - MS. BAGBY AP BIOLOGYbagbyapbio.weebly.com/uploads/1/3/6/5/13650221/1.19_cw_2017_bla… · Adapted by C. Hollinger 2012, revised 2015, 2017 from AP

LabInvestigation3:BLAST

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Name(s):__________________________________Date:_______________Period:_______EssentialQuestion:Howcanbioinformaticsbeusedasatooltodetermineevolutionaryrelationshipsandtobetterunderstandgeneticdiseases?LearningObjectives:

• Tocreatecladogramsthatdepictevolutionaryrelationships• Toanalyzebiologicaldatawithasophisticatedbioinformaticsonlinetool• Tousecladogramsandbioinformaticstoolstoaskotherquestionsofyourownandtotestyour

abilitytoapplyconceptsyouknowrelatingtogeneticsandevolutionBACKGROUNDINFORMATION:Bioinformaticsisaninterdisciplinaryfieldthatcombinescomputerscience,statistics,mathematics,andengineeringtoanalyzeandinterpretbiologicaldata,specificallyinthefieldofgenomics.CommonusesincludeidentifyinggenesandDNAnucleotidesequencestodetermineevolutionaryrelationshipsamongorganisms.BLASTstandsforBasicLocalAlignmentSearchToolanditisdesignedtosearchnucleotideandproteindatabases.Thesequencesmustbeinaspecialformattooptimizeyoursearch.AnyoneintheworldwithacomputercanuseBLAST.YouwillbeusingBLASTn,whichisonlyusedtosearchfornucleotides.Itsearchesformatchesinthesequencebetweenyourgeneofinterest(thegeneyouupload)andmillionsofnucleotidesequencesinitsdatabase.IttakesyourDNAsequenceandbreaksindowninto“smallerwords”madeupof11nucleotidesandthencomparesthemtothoseinthedatabase.Thenitlooksformoresimilar“words”orsequencesadjacenttothefirstoneuntilit“reads”theentireDNAsequenceyouuploadedandhasfoundthemostsimilarmatches,oralignments.Whyisthisinformationimportanttoknow?Beingabletoidentifythepreciselocationandsequenceofhumangenesallowsustobetterunderstandgeneticdiseases.Inaddition,learningaboutthesequenceofgenesinotherspecieshelpsusunderstandevolutionaryrelationshipsamongorganisms.Manyofyourgenesareidenticalorsimilar,tothosefoundinotherspecies. Youwillusetheinformationtoconstructacladogram.Acladogram(alsocalledaphylogenetictree)isavisualizationoftheevolutionaryrelatednessofspecies. Historically,onlyphysicalcharacteristicswereusedtocreatecladograms.Todaymoderncladisticsalsoreliesheavilyongeneticsimilaritiesand/ordifferences.Forexample,theDNAofchimpanzeesandhumansis96-98%thesame,dependingonhowit’scalculated.Thiswouldplacechimpanzeesandhumansveryclosetogetheronacladogram.Humansandfruitfliesshareabout60%ofthesamegenes.Thiswouldplacethemfartherapartonacladogram.

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LabInvestigation3:BLAST

AdaptedbyC.Hollinger2012,revised2015,2017fromAP©BiologyInvestigativeLabs:AnInquiry-BasedApproach.Copyright2012,TheCollegeBoard.https://www.collegeboard.org

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PROCEDURE:

STEP1:UsingFigure4.FossilCladogram,formaninitialhypothesisastowhereyouthinkthefossilspecimenshouldbeplacedonthecladogrambasedonthemorphology(formandstructureofanorganism)youmadeearlier.Writeyourhypothesistotherightofthecladogrambelow.

RemembertodrawwhereyouthinkthefossilspecimenshouldbeonthecladograminFigure4.STEP2:Usethegenesequencehandoutprovidedbyyourteacherandchooseoneofthefossilgenes.Highlightandcopyyourgeneofinterest.

ReadtheparagraphsatthebottomofpageS44.ThisisanenlargedphotoofFigure3.ThisfossilspecimenwasfoundintheLiaoningProvinceinChina.Itisanewlydiscoveredspecies.(Remember:DNAnucleotidesequenceswereextractedfromsofttissueinthisfossil.)YourtaskistouseBLASTntoanalyzethreegenesanddeterminetheplacewherethefossilspeciesismostlikelytobeplacedonthecladogramonpageS45.

Source:AmericanMuseumofNaturalHistory,MickEllison

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LabInvestigation3:BLAST

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STEP3:Uploadthegenesequencebydoingthefollowing:a. GototheBLASThomepage(https://blast.ncbi.nlm.nih.gov/Blast.cgi).Beginbyclickingonthe

NucleotideBlastbox.

Thisisthenextpageyou’llsee.

b. PasteyourgeneofinterestintotheEnterQuerySequencebox.

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LabInvestigation3:BLAST

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*Besuretoaddajobtitlesoyouknowwhichgenenucleotidesequenceyouuploaded.

c. Scrolldowntothebottomofthepage.ClickontheBLASTbutton.

STEP4:Bepatient.Itmaytakeupto60secondstocompletetheBLASTnsearch.You’llseetheresultsin2forms–asadiagram,whichyouseebelow,andatable.

a. ClickonGraphicSummarytoseetheTableofyourBLASTnresults.

b. ViewtheTableofyourBLASTnresults:Thelistofspeciesthatappearsinthissectionarethosewithsequencesidenticaltoormostsimilar,tothegeneyouuploaded.ThesequencesbecomeLESSsimilarareyougodownthelist.

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LabInvestigation3:BLAST

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c. Clickononeofthespecieslistedtofindoutmorespecificinformation,includingtheclassificationscheme(scientificname),theproteinthatthesequencecodesfor,andthesequenceofbasesthatappeartoalignwithyourgeneofinterest.Copythescientificnameofthatspeciesandpasteitintoagooglesearchtofindoutthecommonname.Seetheexamplebelow.

d. Youmustclickontheboxofthespeciesyouwanttousetocreatethe“DistanceTreeofResults.”

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LabInvestigation3:BLAST

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e. ScrollbacktothetopofthepageandclickonDistancetreeofresults.Watchwhathappens!

THISisacladogramofyourresults.Thismightnotmeanmuchtoyourightnow,butit’sastartingpoint.Thiscladogramshowsthespecieswithsimilarsequencestothegeneyouuploaded(geneofinterest),accordingtohowcloselytheirmatchedgenealignswithyourgene.(Seebelow.)

Takeacloserlookatthelegendontherighthand-sideofthepage.Thearrowpointstoyourgene.

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LabInvestigation3:BLAST

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f. ModifytheviewofthecladogrambychangingtheMaxSeqDifferencefrom0.75to0.15.Thiswillgiveyouamorecomprehensiveoverviewoftheevolutionaryrelationships.Usethelegend

todetermineevolutionaryrelationships.Thegeneyouuploadedishighlightedinyellow.

AnalyzingResults(pageS48):AsyoucollectinformationfromBLASTnsearchesforeachofthegenefiles,thinkaboutyouroriginalhypothesisandwhetherthedatasupportyouroriginalplacementofthefossilspeciesonthecladogramonpageS45.Themoresimilargenes2specieshaveincommon,themorerecenttheircommonancestorandthecloserthe2specieswillbelocatedonacladogram.ForeachBLASTnquery,considerthethreecriterialistedonpageS48foreachofthegenefilesbeforeansweringthefollowingquestions.

1. WhatspeciesintheBLASTnresultshasthemostsimilargenesequencestothegeneofinterest?

Firstspecies:

Secondspecies:

Thirdspecies:

2. Whereisthatspecieslocatedonyourcladogram?Firstspecies:Secondspecies:Thirdspecies:

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LabInvestigation3:BLAST

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3. Howsimilaristhegenesequencetoyourgeneofinterest?

Firstgene:Secondgene:Thirdgene:

4. Namethespeciesthathasthenextmostsimilargenesequencetothegeneof

interest.Includeboththescientificnameandthecommonname.EVALUATINGRESULTS(pageS49):Compareanddiscussyourcladogramwithyourclassmates.Doeveryone’sresultsagreewiththeplacementofthefossilspecimenonyourcladogram?Ifthereisdisagreement,whatisthebasisforit?Identifyotherevolutionarydatathatcouldbecollectedfromthefossilspecimentohelpproperlyidentifyitsevolutionaryhistory.DESIGNANDCONDUCTYOUROWNINVESTIGATION:ExampleProcedureYouwilluseBLASTtofindyourowngenesofinteresttoinvestigate.GotopageS50toreadthelistofquestionsandthetablewithsuggestedgenesforhumanproteinsthatyoucouldinvestigate.Pickoneinthetableorfindadifferentone.Forexample:

a. Gotohttps://www.ncbi.nlm.nih.gov/gene.Typehumaninthesearchboxtofindgenesequencesforthousandsofhumanproteins.Followtheinstructionsbelow.

b. IfyouwanttofindoneofthegeneslistedinthetableonpageS50,youcanadd

thenametoyoursearch.Theexamplebelowshowstheresultsofasearchusingonly“human”asthecriteria.Youwillseealonglistofhumanproteins.Chooseaprotein.

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LabInvestigation3:BLAST

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c. ThisexampleshowsasearchforthegenesequenceofHLA-B,anMHCprotein.ClickonHLA-B.Anewpagewillopen.

d. Scrolldownthepageuntilyouseethis.ClickonFASTA.(PronouncedasFast-AY)

e. Thisisjustasnapshotofyourresults.YouneedtoscrolldowntoviewtheentirenucleotidesequenceforHLA-B.

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LabInvestigation3:BLAST

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f. CopytheentiregenesequenceandthengobacktotheBLASTnhomepage.Pasteitintothe“EnterQuerySequence”box,clickBLASTandfollowthesameprocedureyouusedtocreateacladogram.

g. Answerthefollowingquestionstothebestofyourabilityasyoulookataspecificgene.

1. Whatisthefunctioninhumansoftheproteinproducedfromthegeneyouchose?

2. Wouldyouexpecttofindthesameproteininotherorganisms?Ifso,whichones?Explainwhy.

3. Isitpossibletofindthesamegeneintwodifferentkindsoforganismsbutnotfindtheproteinthatisproducedfromthatgene?Explainyouranswer.

4. Ifyoufoundthesameproteininallorganismsyoutest,whatdoesthissuggestabouttheevolutionofthisgeneinthehistoryoflifeoneearth?Justifyyouranswerwithevidence.


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