Biology12 Ms.KimLesson8:BiotechnologyPt.1

Jig-Saw

MainPoint NotesBiologicalToolsandTechniques

• Geneticengineeringistheintentionalalternationofagenomebysubstitutingorintroducing

newgeneticmaterial• Usingvarioustools,molecularbiologistscanCUT,JOIN,andREPLICATEDNA• RECOMBINANTDNAarefragmentsofDNAcomposedofsequencesoriginatingfromatleast

twodifferentsources

RestrictionEnzymes(RestrictionEndonuclease)

Whatarerestrictionenzymesandwhatdotheydo?Restrictionenzymes=molecularscissorsthatcancutDNAataspecificbase-pairsequence

• Recognizespecificsequenceofnucleotides=recognitionsiteWhatisapalindromicsequence?Giveoneexample.Palindromicsequence=bothstrandshavethesamebasesequencewhenreadinthe5’to3’direction

• E.g.EcoRIàG’AATTC/CTTAA’G

HowdorestrictionenzymescutDNA?

• HydrolysisreactiondisruptsphosphodiesterbondsbetweenGandAnucleotides• HydrogenbondsofcomplementaryBPsbetweencellsdisrupted

Whatisthedifferencebetweenstickyendsandbluntends?Giveanexampleeach.

• DependsonwherethecutsoccurStickyends=fragmentendofaDNAmoleculewithshortsingle-strandedoverhangs

• E.g.EcoRIàG’AATTC/CTTAA’GBluntEnds=fragmentendsofaDNAmoleculethatarefullybasepaired

• E.g.SmaIàCCC’GGG/GGG’CCCFromwherearerestrictionenzymesisolatedfromandwhataretheynamedafter?

• Isolatedandpurifiedfrombacteria• Namedafterbacteriafromwhichtheyoriginate

Endonucleasesoftenrecognition4-8basepairsequences.Whatdoesthisinfer.

• Relativelylowfrequencyofcutsincomparisontoa2BPrecognitionsite• E.g.probabilityoffindinga6BPsequenceis4x4x4x4x4x4,onceinevery4096nucleotides• Probabilityoffindinga2BPsequencenexttoeachotheris4x4=onceevery16nucleotides• ImportantingeneticengineeringàifwanttocutapieceofDNAthatincludesawholegene

o Ifcutsarefrequent,genemaybecutandisolatedWhatisDNAligaseandwhatisitsfunction?DNAligase=EnzymeusedforjoiningcutstrandsofDNAtogether(condensationreaction)

Biology12 Ms.KimPlasmidsTransformation

Whatareplasmids?Plasmids=smallcircular,double-strandedDNAmoleculeslackingaproteincoatthatnaturallyexistinthecytoplasmofmanytypesofbacteriaWhatsomeexamplesofthebenefitstheyprovidebacterialcells?

• Genesthatexpressproteinsabletoconferantibioticresistance• Protectbacteriabycarryinggenesforresistancetotoxicheavymetals• Carryplasmidpossessinggenesthatenablebacteriatobreakdownherbicides,industrial

chemicals,orcomponentsofpetroleum

Whatisacopynumberandwhatdoesitrepresent?Copynumber=numberofcopiesofaparticularplasmidfoundinabacterialcell

• Higherthecopynumber,higherthenumberofindividualplasmidsinahostbacterialcell• Morecopies=moreproteinsynthesizedb/clargernumberofgenecopiescarriedbyplasmid• E.g.morecopiesofantibiotic-resistancegene=higherresistancetoantibiotic

Brieflydescribethestepsusedtointroduceaforeigngeneintoaplasmid.Drawadiagram.

1. PlasmidDNAiscutopenat_______RErecognitionsite,producing______ends

• Genefragmentistobeinsertedisexcisedfromsource

2. DNAligasejoinsfragmentandplasmidonincubation

3. Plasmidisintroducedintobacterialcellviatransformation

Whatisatransformation?Transformation=theintroductionofforeignDNA,usuallybyaplasmidorvirus,intoabacterialcell

• Bacteriumthathastakeninaforeignplasmid=being“transformed”

Whatisthedifferencebetweenavector,hostcell,andcompetentcell?

• Plasmidscanbeusedasvectorstocarryadesiredgeneintoahostcell• Competentcell=ifbacteriumreadilytakesupforeignDNA

Mostbacterialcellsarenotnaturallycompetent.Brieflydescribethemethodusedallowingplasmidstoenterthecell.

• Placebacteriainsolutionofcalciumchlorideandrecombinantplasmidsinanice-waterbath• Asbacteriacools,calciumchloridestabilizesthephosphateionsonthephospholipidbilayerof

thecellmembrane• CaCl2solutionthenheatedquicklyandre-cooled• Suddenchangeofcoldtohotmomentarilydisruptsmembrane,allowingplasmidstoenter• Cellsthenkeptat37oCforaperiodoftimetostabilizeandgrow

Biology12 Ms.Kim

GelElectrophoresis

Whatisgelelectrophoresis?Gelelectrophoresis=separationofchargedmoleculesbasedonsizebysortingthroughagelmeshwork

WhatpropertiesofDNAareinvolvedwiththisprocess?Explain.

• DNAfragmentsmustbeseparatedàGEtakesadvantageofchemicalandphysicalproperties

• Samecharge-to-massratio1. Charge2. Molecularmass

Whathouseholditemcanbeusedasananalogyforgelelectrophoresis?Explain.

• MolecularsieveàDNAhasbeencleavedintodifferentsizedfragments

Whatdoesthegelusuallyconsistof?

• Gelusuallyconsistsofabuffercontainingelectrolytesandagarose,orpolyacrylamide(artificialpolymertocreategel)

HowdoessizeandchargeaffectthemovementofDNAthroughthegel?Drawadiagramtosupportyouranswer.

1. DNAisnegativelychargedàeachnucleotidepossessesaphosphategroup(netcharge-1)§ Usingadirectcharge,negativechargeplacedatoneend(wells)àpositive

chargeattheotherendàelectrolytesolutionconveysthecurrent§ -DNAwillmigratetowards+electrode§ Shorterthefragments,fasterthemigration

2. Molecularmassofeachnucleotideisrelativelyconstant(purinesandpyrimidinesbalanceout)§ Shorterthefragment,fasteritwilltravelthroughthegelmeshwork

Biology12 Ms.Kim

WhatisthemostcommonlyusedstaintomakeDNAvisible?

• Ethidiumbromideàcarcinogenic,flatmoleculethatinsertsitselfamongtherungsoftheladderofDNAandfluorescesunderUVlight

Questions:

1. CreateaflowchartdiagramtoshowhowapieceofDNAcontainingatargetgeneisexcised,combinedwithaplasmid,andthenexpressedbyahostcell.

2. HindIIIandSmaIareusedtocutupsamplesofalargepieceofDNA.

Whichenzymewouldyouexpecttoproducealargernumberoffragments?Explain.

• HindIIItohavemorefragmentsàshorterrecognitionsequence• Longertherecognitionsequenceàmorespecificitwillbe• E.g.2BPàprobabilityoffindingitis1in4x4(16)à6%• 8BPà1in4^8(65536)à0.001%

3. Theplasmidbelowwasdigestedusingdifferentrestrictionenzymes

whosesiteshavebeenmapped.Theplasmidis7896basepairslongandcontainsagene(ingreen)thatconferstoantibioticresistancetoacertainmedication.

a) Determinethesizeandnumberoffragmentsthatwouldbe

producediftheplasmidwasdigestedwiththefollowingenzymes:

I) EcoRI• 2fragmentsà1236and6660BP

II) BamHI• 2fragmentsà1399and6497BP

III) HindIII• 3fragmentsà429,1071,6396BP

IV) EcoRIandHindIII• 5fragmentsà372,429,1071,1236,4788BP

V) EcoRI,HindIII,andBAMHI• 7fragmentsà372,429,1071,1399,2333,1236

a. Whichcutwouldinactivatetheantibioticresistance?Why?• BamHIwouldinactivatetheantibioticresistancebecauseitinterruptsthegene