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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