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  • 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
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Lab Investigation 3: BLAST Adapted by C. Hollinger 2012, revised 2015, 2017 from AP © Biology Investigative Labs: An Inquiry-Based Approach. Copyright 2012, The College Board. https://www.collegeboard.org 1 Name(s): __________________________________ Date: _______________ Period: _______ Essential Question: How can bioinformatics be used as a tool to determine evolutionary relationships and to better understand genetic diseases? Learning Objectives: • To create cladograms that depict evolutionary relationships • To analyze biological data with a sophisticated bioinformatics online tool • To use cladograms and bioinformatics tools to ask other questions of your own and to test your ability to apply concepts you know relating to genetics and evolution BACKGROUND INFORMATION: Bioinformatics is an interdisciplinary field that combines computer science, statistics, mathematics, and engineering to analyze and interpret biological data, specifically in the field of genomics. Common uses include identifying genes and DNA nucleotide sequences to determine evolutionary relationships among organisms. BLAST stands for Basic Local Alignment Search Tool and it is designed to search nucleotide and protein databases. The sequences must be in a special format to optimize your search. Anyone in the world with a computer can use BLAST. You will be using BLASTn, which is only used to search for nucleotides. It searches for matches in the sequence between your gene of interest (the gene you upload) and millions of nucleotide sequences in its database. It takes your DNA sequence and breaks in down into “smaller words” made up of 11 nucleotides and then compares them to those in the database. Then it looks for more similar “words” or sequences adjacent to the first one until it “reads” the entire DNA sequence you uploaded and has found the most similar matches, or alignments. Why is this information important to know? Being able to identify the precise location and sequence of human genes allows us to better understand genetic diseases. In addition, learning about the sequence of genes in other species helps us understand evolutionary relationships among organisms. Many of your genes are identical or similar, to those found in other species. You will use the information to construct a cladogram. A cladogram (also called a phylogenetic tree) is a visualization of the evolutionary relatedness of species. Historically, only physical characteristics were used to create cladograms. Today modern cladistics also relies heavily on genetic similarities and/or differences. For example, the DNA of chimpanzees and humans is 96-98% the same, depending on how it’s calculated. This would place chimpanzees and humans very close together on a cladogram. Humans and fruit flies share about 60% of the same genes. This would place them farther apart on a cladogram.

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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

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

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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.

Page 2: 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

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

Page 3: 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

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

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

NucleotideBlastbox.

Thisisthenextpageyou’llsee.

b. PasteyourgeneofinterestintotheEnterQuerySequencebox.

Page 4: 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

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

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*Besuretoaddajobtitlesoyouknowwhichgenenucleotidesequenceyouuploaded.

c. Scrolldowntothebottomofthepage.ClickontheBLASTbutton.

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

a. ClickonGraphicSummarytoseetheTableofyourBLASTnresults.

b. ViewtheTableofyourBLASTnresults:Thelistofspeciesthatappearsinthissectionarethosewithsequencesidenticaltoormostsimilar,tothegeneyouuploaded.ThesequencesbecomeLESSsimilarareyougodownthelist.

Page 5: 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

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

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

d. Youmustclickontheboxofthespeciesyouwanttousetocreatethe“DistanceTreeofResults.”

Page 6: 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

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

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e. ScrollbacktothetopofthepageandclickonDistancetreeofresults.Watchwhathappens!

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

Takeacloserlookatthelegendontherighthand-sideofthepage.Thearrowpointstoyourgene.

Page 7: 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

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

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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:

Page 8: 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

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

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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.

Page 9: 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

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

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

d. Scrolldownthepageuntilyouseethis.ClickonFASTA.(PronouncedasFast-AY)

e. Thisisjustasnapshotofyourresults.YouneedtoscrolldowntoviewtheentirenucleotidesequenceforHLA-B.

Page 10: 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

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

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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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