AP Biology Day 34 Monday, November 14, 2016
Essen%alknowledgestandards• 3.A.1:DNA,andinsomecasesRNA,istheprimarysourceofheritableinforma%on
• 3.A.1.e:Gene%cengineeringtechniquescanmanipulatetheheritableinforma%onofDNAand,inspecialcases,RNA– Electrophoresis– Plasmid-basedtransforma%on– Restric%onenzymeanalysisofDNA– PolymeraseChainReac%on(PCR)
FLT• Iwillbeableto:– describethefunc%onofrestric%onenzymesandexplainhowtheyareusedinrecombinantDNAtechnology
– Outlinetheproceduresforcloningaeukaryo%cgeneinabacterialplasmid
– Explainhowgelelectrophoresisisusedtoanalyzenucleicacidsandtodis%nguishbetweentwoallelesofagene
• Bycomple1ngCh.20LectureNotes
Ch.20:Biotechnology
Whatisbiotechnology?• Biotechnologyuseslivingorganismsortheirgene%ccontenttomakeusefulproducts,suchasGMOs
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Overview• Sequencingofthehumangenomewascompletedby2007.
• DNAsequencinghasdependedonadvancesintechnology,star%ngwithmakingrecombinantDNA.
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I.RecombinantDNAA. Def.–Nucleo4de
sequencesfromtwodifferentsourcesarecombinedinvitrointothesamemolecule
– Invitroreferstoaprocessthatoccursoutsideofabody/cell(inanar%ficialmedium)
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I.RecombinantDNAB. Gene%cengineering=directmanipula4onof
genesviabiotechnology
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I.RecombinantDNAC. Biotechnology=manipulateslivingorganismsor
theirgene4ccontenttomakeusefulproducts,suchasGMOs
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II.StrategiesofGeneManipula%onA. RecombinantDNAandCloningB. AmplifyingDNAinVitro
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A.RecombinantDNAandCloning1. Plasmids2. GeneCloning3. Eukaryo%cGeneCloninginaBacterialPlasmid
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1.Plasmid– Plasmids=smallcircularDNAmoleculesthatreplicate
separatelyfromthebacterialchromosome.– Thismakesthemusefultoscien%ststoclone,transfer,
andmanipulategenes!– TheycaninsertpiecesofDNA(genes)intoaplasmid,
andthenhavethebacteriumreplicatethegenerapidly!
– Clonedgenesareusefulformakingcopiesofapar%culargeneandproducingaproteinproduct.
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RecombinantDNAPlasmid&GeneCloning
DNAofchromosome
Cellcontaininggeneofinterest
Geneinsertedintoplasmid
Plasmidputintobacterialcell
RecombinantDNA(plasmid)
Recombinantbacterium
Bacterialchromosome
Bacterium
Geneofinterest
Hostcellgrowninculturetoformacloneofcellscontainingthe“cloned”geneofinterest
Plasmid
GeneofInterest
Proteinexpressedbygeneofinterest
Basicresearchandvariousapplica4ons
Copiesofgene Proteinharvested
Basicresearchongene
Basicresearchonprotein
Geneforpestresistanceinsertedintoplants
Geneusedtoalterbacteriaforcleaninguptoxicwaste
ProteindissolvesbloodclotsinheartaQacktherapy
Humangrowthhor-monetreatsstuntedgrowth
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2.GeneCloninga. Restric%onEnzymesb. Restric%onSitesc. Restric%onFragmentsd. S%ckyEndse. DNAligasef. CloningVector
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Wheredowegetrestric%onenzymes?
• Bacteriacellsproducerestric%onenzymesasadefenseagainstbacteriophages
• BamHI:Bacillusamyloliquefaciens• HindIII:Haemophilusinfluenza
2.GeneCloninga. Restric%onEnzymes(RE’s)=cutDNAmoleculesat
specificDNAsequencescalledrestric4onsites.b. Restric%onSites=specificbasepairsequence
recognizedbyrestric4onenzymes
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Whatisarestric%onenzyme?
• AnenzymethatcutsDNAataspecificsite(sequence)
BluntEnds
“Sticky” Ends
2.GeneCloninga. Restric%onEnzymes(RE’s)b. Restric%onSites
– Specificenzymescutatspecificsequences
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Whatisapalindrome?• Eye• Racecar• Neveroddoreven
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Howdorestric%onenzymescut?
• CutatPalindromes:thecomplimentaryDNAisapalindrometotheDNAstrand
• Palindromes:Sameforwards/backward:• Anutforajaroftuna• Eva,canIstabbatsinacave?
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2.GeneCloningc. Restric%onFragments
– Arestric4onenzymeusuallymakesmanycuts,yieldingrestric4onfragments.
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2.GeneCloningd. S%ckyEnds
– BoththeplasmidDNAandgeneofinterestshouldbecutwiththesamerestric4onenzyme(s)inordertocreatecomplementarys4ckyends
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2.GeneCloninge. DNAligasea. Sealsthe(covalent)bondsbetweenrestric4on
fragments
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2.GeneCloningf. CloningVector• Originalplasmid=thecloningvector=aDNAmoleculethatcancarryforeignDNAintoahostcellandreplicatethere.
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Plasmids
– Cloningvectorplasmid• Engineeredtoreplicateinhighnumberswithinbacteria
– Expressionvectorplasmid• Carriesthegeneofinterestinaspecificloca%onthatallowsthebacteriatoexpressthegene
CloningVectorplasmidContains:1. Originofreplica%on
2. Selectablemarkergene
3. Mul%plecloningsite
ToMakeRecombinantDNA
Restric4onsite
DNA
S4ckyend
Restric4onenzymecutssugar-phosphatebackbones.
5ʹ3ʹ
3ʹ5ʹ
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Onepossiblecombina4on
RecombinantDNAmolecule
DNAligasesealsstrands.
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DNAfragmentaddedfromanothermoleculecutbysameenzyme.Basepairingoccurs.
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CloningRecombinantGenes Bacterialcell
Bacterialplasmid
lacZgene
Hummingbirdcell
Geneofinterest
HummingbirdDNAfragments
Restric4onsite S4cky
endsampRgene
TECHNIQUE
Recombinantplasmids
Nonrecombinantplasmid
Bacteriacarryingplasmids
RESULTS
Colonycarryingnon-recombinantplasmidwithintactlacZgene
Oneofmanybacterialclones
ColonycarryingrecombinantplasmidwithdisruptedlacZgene
3.Eukaryo%cGeneCloninginaBacterialPlasmid
a. Isola%onofDNAandplasmidb. Treatmentwiththesamerestric%onenzymec. MixforeignDNAwithplasmidsd. Addi%onofDNAligasee. Introduc%onofnewplasmidintobacterialcells
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ToMakeRecombinantDNA
Restric4onsite
DNA
S4ckyend
Restric4onenzymecutssugar-phosphatebackbones.
5ʹ3ʹ
3ʹ5ʹ
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Onepossiblecombina4on
RecombinantDNAmolecule
DNAligasesealsstrands.
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DNAfragmentaddedfromanothermoleculecutbysameenzyme.Basepairingoccurs.
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CloningRecombinantGenes Bacterialcell
Bacterialplasmid
lacZgene
Hummingbirdcell
Geneofinterest
HummingbirdDNAfragments
Restric4onsite S4cky
endsampRgene
TECHNIQUE
Recombinantplasmids
Nonrecombinantplasmid
Bacteriacarryingplasmids
RESULTS
Colonycarryingnon-recombinantplasmidwithintactlacZgene
Oneofmanybacterialclones
ColonycarryingrecombinantplasmidwithdisruptedlacZgene
B.AmplifyingDNAinVitro1. PolymeraseChainReac%on• =PCR=producesmanycopiesofaspecificsegmentofDNA
• Eachcycleheats,cools,andreplicatesDNAtoproduceiden4calDNAmoleculesatanexponen4alrate
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PCR 5ʹ
GenomicDNA
TECHNIQUE
Cycle1yields2
molecules
Denatura4on
Annealing
Extension
Cycle2yields4
molecules
Cycle3yields8
molecules;2molecules(inwhiteboxes)
matchtargetsequence
Targetsequence
Primers
Newnucleo-4des
3ʹ
3ʹ
3ʹ
3ʹ
5ʹ
5ʹ
5ʹ1
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• DNAcloningallowsresearchersto– Comparegenesandallelesbetweenindividuals.– Locategeneexpressioninabody.– Determinetheroleofageneinanorganism.
• SeveraltechniquesareusedtoanalyzetheDNAofgenes:GelElectrophoresis,restric1onfragmentanalysis,Southernblogng,DNAsequencing.
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III.DNATechnologyandtheStudyofGenesA. GelElectrophoresisB. Restric%onFragmentAnalysisC. Gene%cEngineering
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A.GelElectrophoresis• Oneindirectmethodofrapidlyanalyzingandcomparinggenomesisgelelectrophoresis.
• Thistechniqueusesagelasamolecularsievetoseparatenucleicacidsorproteinsbysize.
• Acurrentisapplied,andchargedmoleculesmovethroughthegelandaresortedinto“bands”bytheirsize
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GelElectrophoresis Mixtureof
DNAmol-eculesofdifferentsizes
Powersource
Powersource
Longermolecules
Shortermolecules
Gel
AnodeCathode
TECHNIQUE
RESULTS
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GelElectrophoresis• Onceyourunagel,youcanvisualizetheDNAbands• YoucanuseaDNAladder/markertodeterminethebasepairlengths
• Wecanthenusethisdatatocreatearestric%onmap
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Example• Let’ssaytheenzymesBglIIandBstEIItogetherproducetwofragmentsof6008and1658basepairs
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EcoRVcutstwice:4729&2937segments
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IfEcoRVandBgIIIareputtogether• Wegetthreefragments:4729,1992,and945bps• SoBglIImusthavemadeacutbetweenthefirstandsecondEcoRYsegments
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Key• Whenmakingarestric%onmap,itsome%meshelpstofirststartwithcutsmadebyonerestric%onenzyme,thenaddingotherenzymesasyougo
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B.Restric%onFragmentAnalysis• =DNAfragmentsproducedbyrestric4onenzymediges4onofaDNAmoleculearesortedbygelelectrophoresis.
• Restric%onfragmentanalysisisusefulforcomparingtwodifferentDNAmolecules,suchastwoallelesforagene.
• Theprocedureisalsousedtopreparepuresamplesofindividualfragments.
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B.Restric%onFragmentAnalysis• RFLP=Restric4onfragmentlengthpolymorphism=differencesinrestric4onfragmentlengthsinindividuals
• Canbeusedasmolecularmarkers– EachindividualwillhaveuniqueDNAandtheirrestric%onfragmentswillbeuniqueaswell
– Byrunningagel,wecancreateaDNAfingerprint,whichareolenusedinforensicscience
– Canalsobeusedasmarkerstoiden%fypar%culargroupsofpeopleatriskforcertaingene%cdisorders
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Restric1onFragmentAnalysis
Normalallele
Sickle-cellallele
Largefragment
(b)Electrophoresisofrestric4onfragmentsfromnormalandsickle-cellalleles
201bp175bp
376bp
(a)DdeIrestric4onsitesinnormalandsickle-cellallelesofβ-globingene
Normalβ-globinallele
Sickle-cellmutantβ-globinallele
DdeI
Largefragment
Largefragment
376bp
201bp175bp
DdeIDdeI
DdeI DdeI DdeI DdeI
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C.Gene%cEngineering2. Stemcells• Astemcellisarela%velyunspecializedcellthatcanreproduceitselfindefinitelyanddifferen%ateintospecializedcellsofoneormoretypes.
• Stemcellsisolatedfromearlyembryosattheblastocyststagearecalledembryonicstemcells;theseareabletodifferen%ateintoallcelltypes.
• Theadultbodyalsohasstemcells,whichreplacenonreproducingspecializedcells.
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Review
Cutbysamerestric4onenzyme,mixed,andligated
DNAfragmentsfromgenomicDNAorcDNAorcopyofDNAobtainedbyPCR
Vector
RecombinantDNAplasmids
Fig.20-UN4
G
AardvarkDNA
Plasmid
5ʹ3ʹ
3ʹTCCATGAATTCTAAAGCGCTTATGAATTCACGGC5ʹAGGTACTTAAGATTTCGCGAATACTTAAGTGCCG
ACTT
AA
Restric4onEnzyme:HindIII-->Restric4onFragments/S4ckyEnds
Pair-Share-Respond1. Ifyouwanttocloneageneusinga
bacterialplasmid,whatstepswouldyoutake?
2. Whymustplasmidsandthegeneofinterestbecutwiththesamerestric4onenzymes?
3. WhatarethethreepartsofPCRandwhatdoesPCRproduce?
4. Explaintheuseofgelelectrophoresis
CW/HW• Finishnotes• FRQ• Lab2AQues%ons
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