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Peter J.

Russell
AmolecularApproach2ndEdition

CHAPTER 8
Recombinant DNA
Technology
editedbyYueWenWangPh.D.
Dept.ofAgronomy,NTU

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Chapter7slide1

DNACloning
1. Thegoalofmolecularcloningislargeamountsofpure
DNAthatcanbefurthermanipulatedandstudied.
2. Summaryoftheprocedure:
a. IsolateDNAfromtheorganism.
b. UserestrictionenzymestocuttheDNA,andligatefragments
intoacloningvector.
c. TransformrecombinantDNAintoahost,whichwillreplicate
theDNA(molecularcloning)andpasscopiestoallprogeny.

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Chapter7slide2

RestrictionEnzymes
1. Restrictionendonucleases(restrictionenzymes)eachrecognizea
specificDNAsequence(restrictionsite),andbreakaphosphodiester
linkagebetweena3carbonandphosphatewithinthatsequence.
2. RestrictionenzymesareusedtocreateDNAfragmentsforcloning,and
toanalyzepositionsofrestrictionsitesinclonedorgenomicDNA.
3. RestrictionenzymesareabacterialdefenseagainstviralDNA.
Restrictionsitesinthebacterialchromosomearemethylated,andthus
protected.
4. Arestrictionenzymehasathreeletternamederivedfromthegenus
andspeciesoftheorganismfromwhichitwasisolated;itisunderlined
oritalicized.Romannumeralsandsometimeslettersdesignatinga
particularbacterialstrainmayfollow.
5. Manyrestrictionsitesarepalindromesof4,6or8basepairs,but
othersarenotcompletelysymmetrical(Figure8.1andTable8.1).
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Chapter7slide3

Fig. 8.1 Restriction site in DNA, showing symmetry of the sequence around the
center point

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

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Chapter7slide4

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Chapter7slide5

6. Allcopiesofachromosomewillcontainthesamerestrictionsites,and
willbecutintoidenticalfragments.
7. Basedonprobability,aspecificshortDNAsequenceoccursmore
frequentlythanalongerone.
a. Ina50%G-Corganismwithrandomdistributionofbases,the
probabilityofaspecificbaseatagivenpositionis 14.
b. Therefore,thefrequencyofaparticularrestrictionsiteis( 14)n,
wherenisthenumberofbasepairsintherecognitionsequence.
8. OnemajorclassofrestrictionenzymesrecognizesandcutsDNA
atspecificsequences.TwotypesofDNAendscanbegenerated
(Figure8.2).
a. Somerestrictionenzymesproducebluntends,wherebothDNA
strandsarecutbetweenthesamebasepairs.
b. Otherscreatesticky(staggered)ends.
9. Stickyendsareusefulincloning,becausecomplementarysequences
hydrogenbond(anneal)andareheldtogethersothatDNAligasecan
covalentlylinkthem(Figure8.3).
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Chapter7slide6

Fig. 8.2 Examples of how restriction enzymes cleave DNA

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Chapter7slide7

Fig. 8.3 Cleavage of DNA by the restriction enzyme EcoRI

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

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Chapter7slide8

CloningVectorsandDNACloning
Animation:DNACloninginaPlasmidVector
1.Severaltypesofcloningvectorshavebeenconstructed,eachwithdifferentmolecularpropertiesand
cloningcapacity.
2.Plasmidcloningvectorsarederivedfromnaturalplasmids,circlesofdsDNAthatincludeorigin
sequences(ori)neededforreplicationinbacterialcells.AnE.coliplasmidvector,forexample,
mustcontainthesefeatures:
a.Anorisequenceforreplication.
b.Aselectablemarker,suchasantibioticresistance.
c.Uniquerestrictionsites,sothataparticularrestrictionenzymecutsonlyonceintheplasmid.Afragmentof
insertDNAcutwiththesameenzymeiscommonlyinsertedintotheuniquerestrictionsite.

3.AnexampleofatypicalE.colicloningvectorispUC19(2,686bp).ThepUC19plasmidfeatures:
a.HighcopynumberinE.coli,withnearlyahundredcopiespercell,providesagoodyieldofclonedDNA.
b.ItsselectablemarkerisampR.
c.Ithasaclusterofuniquerestrictionsites,calledthepolylinker(multiplecloningsite).
d.ThepolylinkerispartofthelacZ(galactosidase)gene.ThepUC19plasmidwillcomplementalacZE.coli,
allowingittobecomelacZ+.WhenDNAisclonedintothepolylinker,lacZisdisrupted,preventing
complementationfromoccurring.
e.Xgal,achromogenicanalogoflactose,turnsbluewhengalactosidaseispresent,andremainswhiteinits
absence,sobluewhitescreeningcanindicatewhichcoloniescontainrecombinantplasmids(Figure8.4).

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Chapter7slide9

Fig. 8.4 The plasmid cloning vector pUC19

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

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Chapter7slide10

4. DNAcanbeinsertedintoacloningvectorbyrestrictiondigestionandthenligation
(Figure8.5).
a. CutpUC19(thevectorplasmid)witharestrictionenzymethathasauniquesiteinthe
polylinker.
b. CuttheDNAtobecloned(insertDNA)withthesameenzyme.
c. MixinsertDNAwithpUC19DNAandallowrandomjoiningoffragmentstooccur.
d. ResultingplasmidsaretransformedintoE.colieitherthroughchemicaltreatmentofthecells
orbyelectroporation.ThecellsaregrownonmediaplatescontainingampicillinandXgal.
e. AmpicillinresistantcoloniesresultfrompUC19sequences.Bluecoloniescontainonlythe
vectorwithitsendsrejoined,whilewhitecoloniesoftencontainpUC19withitslacZgene
inactivatedbyinsertDNA.
f. Ifthe5phosphatesofvectorDNAareremovedbyalkalinephosphatase,DNAligasewillnot
rejointheirends,andfewerbluecolonieswillresult.

5. Ifoneenzymeisusedincloning,twoorientationsarepossiblefortheinsert.Atwo
enzymestrategyresultsinonlyoneorientation.
6. Manyplasmidcloningvectorsareavailable,withfeaturesincludingdifferentarraysof
uniquerestrictionsitesinthepolylinker,andphagepromoters(e.g.,T7,T3,SP6)that
canbeusedtocontroltranscriptionoftheclonedDNA.
7. Plasmidcloningvectorsareavailableformanyprokaryoticandeukaryoticorganisms.In
somecasestheplasmidsareunabletoreplicate,butaremaintainedbecausethey
integrateintothegenome.
8. SizeoftheinsertDNAislimitedinplasmidcloningvectors,andplasmidscarryingmore
than510kbareoftenunstable.
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Chapter7slide11

Fig. 8.5 Insertion of a piece of DNA into the plasmid cloning vector pUC19

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

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Chapter7slide12

ShuttleVectors
1. Acloningvectorcapableofreplicatingintwoor
moretypesoforganism(e.g.,E.coliandyeast)is
calledashuttlevector.Shuttlevectorsmay
replicateautonomouslyinbothhosts,orintegrate
intothehostgenome

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Chapter7slide13

ExpressionVectors
1. Expressionvectorshavesequencestoallowtranscription
andtranslationoftheclonedgene(s).Pharmaceuticals
producedbybiotechnologyareanexample.
2. Plasmidcloningvectorsaremodifiedtoinclude:
a. Promoterandtranscriptionterminatorifneeded,suitabletothe
hostorganism.
b. Anymodificationsneededtocrossprokaryotic/eukaryotic
boundary(e.g.,ShineDelgarnosequenceisaddedfortranslation
ofeukaryoticsequencesinE.coli).

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Chapter7slide14

ArtificialChromosomes
1. Cloningvectorsthatcanaccommodateverylarge
piecesofDNAproducemoleculesresembling
smallchromosomes.TwoexamplesareYACs
andBACs.

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Chapter7slide15

YeastArtificialChromosomes(YACs)
1.

YACvectorsfunctionasartificialchromosomesinyeast.Theirfeaturesinclude
(Figure8.6)
a.

Linearstructurewithayeasttelomere(TEL)ateachend.

b.

Ayeastcentromeresequence(CEN).

c.

Amarkergeneoneacharmthatisselectableinyeast.(e.g.TRP1andURA3)

d.

Ayeastoriginofreplicationknownasautonomousreplicatingsequence
(ARS).

e.

UniquerestrictionsitesforinsertingforeignDNA.

2.

SeveralhundredkbofinsertDNAcanbeclonedinaYAC.However,frequent
RNArearrangementsinthehostmakeYACsunsuitableforgenomesequencing.

3.

YACclonesaremadeby:
a.

PropagatingtheDNAinE.coliasacircularplasmidwithtelomeresendtoend.

b.

Cutwithrestrictionenzymesinmultiplecloningsiteandanotherbetweenthetwo
TELs,generatingtwoarms.

c.

LigatinglonginsertDNAfragmentwiththetwoarms.

d.

Transformingintoyeast.

e.

Selectingformarkers(e.g.,TRP1andURA3)toensurethatbotharmsarepresent.
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Chapter7slide16

Fig. 8.6 Example of a yeast artificial chromosome (YAC) cloning vector

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

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Chapter7slide17

BacterialArtificialChromosomes(BACs)
1. BACsareusedforcloningfragmentsuptoabout200kb
inE.coli.BACvectorscontain:
a.theoriofanE.coliplasmidcalledtheFfactor.
b.Amultiplecloningsites.
c.Aselectablemarker.
d.Otherfeatures,bellsandwhistles.

2.BACcanbehandleslikeregularbacterialplasmids,but
theFfactororikeepscopynumberatoneBACmolecule
percell.
3. BACsdonotundergorearrangementsinthehost.
4. BACsarecommonlyusedtostudygeneregulationin
vertebrates.
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Chapter7slide18

iActivity:BuildingABetterBeer?

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Chapter7slide19

RecombinantDNALibraries
1. Itissometimesusefultohaveagenomiclibrary,a
collectionofclonescontainingatleastonecopy
ofeveryDNAsequenceinagenome.Genomic
librariesareavailableformanyorganisms.
2. Chromosomelibrariesarecollectionsofcloned
fragmentsofindividualchromosomes.
3. ComplementaryDNA(cDNA)librariesare
clonedcollectionsofDNAcopiedfromacells
mRNA.
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Chapter7slide20

GenomicLibraries
1.

AgenomiclibraryisusedtoisolateandstudyaclonedDNA
containingasequenceofinterest.Genomiclibrariesofeukaryotic
DNAareconstructedbydigestinggenomicDNAwitharestriction
enzyme,andligatingintoavectorthatcanaccommodatethegenome
inamanageablenumberofclones.

2.

Therearethreegeneralwaystoproducegenomiclibraries:
a.

b.

Completedigestionwithrestrictionenzyme,cleaveatallrelevant
restrictionsites.Thishasdrawback:
1)

Genescontainningoneormoresitesfortherestrictionenzyme
willbeclonedintotwoormorepieces.

2)

Toscreentheentiregenome,averylargenumberofclones
wouldhavetobeexamined,becauseinsertDNAsizeisrelative
small.

LongerDNAfragmentscanbegeneratedwithmechanicalsheering
(e.gpassagethroughasyringeneedle)ratherthanrestrictionenzyme
cutting.Adisadvantageistheabsenceofuniformends,require
enzymaticmodificationbeforeinsertionintoaclonningvector.
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Chapter7slide21

c.

3.

Partialdigestionwitharestrictionenzymeiscontrolledsothatitcuts
onlysomeoftheavailablesites.Ideally,thisresultsincloninga
populationofoverlappingfragmentsrepresentingtheentiregenome
(Figure8.7)
1)

PartiallydigestedDNAmoleculesinacertainsizerangeare
selectedbydensitygradientcentrifugationoragarosegel
electrophoresis.

2)

DNAfragmentswithstickyendsfromrestrictiondigestioncan
becloneddirectly.Sometimestwoenzymeswithcompatible
stickyendsareused(e.g.BamHIandSau3A),creatingahybrid
recognitionsite.

3)

Genomicsequencesarenotequallyrepresentedinthelibraries,
because:
a)

RegionsofDNAwithrelevantrestrictionsitesveryclose
togetherorveryfarapartareremovedatthesizeselection.

b)

SomeregionsofeukaryoticDNApreventvectorreplication
inE.coli,andsoareeliminatedfromthelibrary.

Thenumberofclonesneededforacompletelibrarycanbecalculated
basedonthesizeofthegenomeandtheaveragesizeofDNA
insertedintothevector.Inpractice,alibraryshouldcontainmany
timesmorethanthecalculatedminimumnumberofclones.
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Chapter7slide22

Fig. 8.7 Use of partial digestion with a restriction enzyme to produce DNA fragments
of appropriate size for constructing a genomic library

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

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Chapter7slide23

Chromosomelibraries
1. Screeningthegenomiclibraryofanorganism
withalargegenomeislaborious.Screeningtime
canbereducedifagenehasbeenlocalizedtoa
chromosome,byexaminingalibrarymadefrom
onlythatchromosome.Human,forexample,
have24differentchromosomelibraries(22
autosomes,XandY).
2. Separatingchromosomesotheymaybe
individuallyclonedisaccomplishedwith
techniquessuchasflowcytometry.
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Chapter7slide24

cDNALibraries
1. AcellsmRNAmoleculescanbecopiedtomakecomplementaryDNA
strands(cDNA)andthecDNAcanthenbecloned,creatingalibrary
representingonlythegenesbeingexpressedinthecellsatthattime.
2. ThecDNAderivesonlyfrommaturemRNA.Intronsarenotpresent.
3. Thepoly(A)tailatthe3endofthemRNAisusefulfor:
a. IsolatingmRNAfromcelllysatesbypassageoveranoligo(dT)
column.
i.ThemRNAspoly(A)tailstickstothepoly(T)attachedtothe
columnsubstrate.
ii.Othermoleculespassthroughthecolumn,butmRNAsare
retained.
iii.mRNAsareelutedwithdecreasingionicstrengthbuffer,resulting
insignificantpurification.
b. PrimingthesynthesisofcDNA,byprovidingaknown5sequence.
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Chapter7slide25

4. SynthesisofcDNAinvolvesthesesteps(Figure8.8):
a. Ashortoligo(dT)primerisused.ItannealstothemRNAs
poly(A)tail,allowingreversetranscriptasetosynthesizecDNA.
ThiscreatesaDNAmRNAhybrid.
b. RNaseHdegradesthemRNAstrand,creatingsmallRNA
fragmentsthatserveasprimers.
c. DNApolymeraseImakesnewDNAfragments,andDNAligase
connectsthenewDNAfragmentstomakeacompletechain.
d. TheresultingcDNAisadoublestrandedcopyofthestarting
mRNA.
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Chapter7slide26

Fig. 8.8 The synthesis of complementary DNA (cDNA) from a polyadenylated mRNA

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

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Chapter7slide27

5. AmethodforcloningcDNAinvolves(Figure8.9):
a. IntroducingrestrictionsitelinkerstotheendsofthecDNAby
bluntendligation.
b. Digestionwiththecognaterestrictionenzymetocreatesticky
ends.
c. MixingcDNAwithvectorDNAcutwiththesamerestriction
enzymeinthepresenceofDNAligase.
d. TransformingintoanE.colihostforcloning.

6.IfthecDNAhassitesforthesamerestrictionenzyme
usedinthepolylinker,thecDNAwillbeclonedinpieces.
Theproblemisavoidedbyusingpolylinkersengineered
withappropriatessDNAoverhangs(stickyends)sothat
restrictiondigestionisunnecessary.
7. Bluntendcloningisalsoused.
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Chapter7slide28

Fig. 8.9 The cloning of cDNA using BamHI linkers

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Chapter7slide29

FindingaSpecificCloneinaLibrary
1. Anumberoftechniqueshavebeendevelopedfor
identifyingthecloneofinterestinacDNA
library.Somearediscussedhere.

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Chapter7slide30

ScreeningacDNAlibrary
1.OnewaytoselectacDNAclonefromthelibraryistodetectaprotein
productitisproducing(Figure8.10).
2.Forproteintobeproduced,anexpressionvectorisneeded,inwhichthe
clonedcDNAisinsertedbetweenapromoterandatranscription
terminator.
3.Labeledantibodiesareusedtodetectthespecificproteininahost
colony.Anarrayofcoloniesistransferredtoamembranefiter,cells
arelysedandtheirproteinsbindtothefilter,whichisincubatedwith
therelevantantibody.
4.Radioactivelylabeledantibodyboundtocoloniesisdetectedbyan
autoradiogram,inwhichthedryfiterisplacedonXrayfilminthedark
foranumberofhours.Colonieswithantibodyboundwillbevisibleas
darkspotsonthefilm.
5.Onceidentified,thecDNAcanbeusedto:
a.Analyzethegenomeofthesameoranotherorganismforhomologous
sequences.
b.IsolatethenucleargeneforthemRNAfromagenomiclibrary.
c.QuantifymRNAsynthesizedfromthegene.

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Chapter7slide31

Fig. 8.10 Screening for specific cDNA plasmids in a cDNA library by using an antibody
probe

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

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Chapter7slide32

ScreeningaGenomicLibrary
1. Finding a specific clone in a plasmid or cosmid genomic library is
similar to screening a cDNA library (Figure 8.11).
a. E. coli cells are transformed with the genomic library, and plated on
selective medium.
b. Colonies produced are replica plated onto a membrane filter on a plate
of selective medium, and cells grow on the filter.
c. Cells are lysed, DNA is denatured and bound tightly to the filter.
d. Filter is incubated with the labeled single-stranded cDNA probe, which
forms hybrids with complementary DNA molecules bound to the filter.
e. Filter is washed and the label is detected by autoradiography for a
radioactive probe, or chemiluminescent or colorimetric assay for
nonradioactive labeling.
f. Clones detected by the probe are then further characterized.

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Chapter7slide33

Fig. 8.11 Using DNA probes to screen plasmid or cosmid genomic libraries for specific
DNA sequences

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Chapter7slide34

IdentifyingGenesinLibrariesby
ComplementationofMutations
1. Well-defined mutants may be used to clone genes by complementation,
in which cloned genes overcome a defect in the mutant.
2. To clone a yeast gene by complementation:
a. A genomic library is made from the wild-type yeast strain in a yeast-E.
coli shuttle vector.
b. The library is transformed into a yeast strain with two mutations, one to
allow selection of transformants, and the other a mutation in the gene
for which the wild-type genes sought The ARGJ gene is an example
(Figure 8.12).
c. Transformed yeast with the wild-type phenotype restored for the gene
sought are selected and their plasmid DNA further characterized.

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Chapter7slide35

Fig. 8.12 Example of cloning a gene by complementation of mutations: the cloning of


the yeast ARG1 gene

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

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Chapter7slide36

IdentifyingSpecificDNASequencesinLibraries
UsingHeterologousProbes

1. It is possible to identify specific genes in a


genomic library using cloned equivalent genes
(heterologous probes) from other organisms,
especially if the gene is highly conserved or the
species are closely related.

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Chapter7slide37

IdentifyingGenesorcDNAsinLibraries
UsingOligonucleotideProbes
1. Synthetic oligonucleotides are useful in probing libraries, sequence data
are available for part of the gene of interest. Knowledge of substitutions
produced by mutation also aids probe selection. Sequences for many
genes are available in GenBank.
2. Using the universal genetic code, the amino acid sequence is used to
design a DNA oligonucleotide probe. Degeneracy in the genetic code
means that a mixture of oligonucleotides must be prepared, each of
which encodes the target protein.
3. The library is probed with the oligonucleotide mixture to detect the gene
of interest. This is a powerful (but not perfect) technique for isolating
specific clones.

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Chapter7slide38

MolecularAnalysisofClonedDNA
1. ClonedDNAisusedinmanytypesof
experiments.Threeexamplesare:
a. Restrictionmapping
b. Southernblotting
c. Northernblotting

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Chapter7slide39

RestrictionMapping
Animation:RestrictionMapping
1. ClonedDNAcanbecutwithrestrictionenzymesandelectrophoresed
onagarosegelsandvisualizedwithethidiumbromide,inordertomap
itsrestrictionsites(Figure8.13).
2. Examplesofusesforrestrictionmaps:
a. SubcloningsectionsofgeneorcDNA.
b. Confirmingresultsofacloningexperiment.
c. ComparingcDNAwithitsgene.
d. Constructingphysicalmapsofchromosomes.
3. TheDNAiscutwithseveraldifferentenzymes,andeachcutisloaded
inalaneofanagarosegel.Electricalcurrentdrivesthenegatively
chargedDNAfragmentsthroughthegel.Smallmoleculesmovemore
quicklythanlargeones,sothefragmentsareseparatedbysize.

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Chapter7slide40

Fig. 8.13 Constructing a restriction map for EcoRI and BamHI in a DNA fragment

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

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Chapter7slide41

4. DNAisstainedwithethidiumbromide,whichfluoresces
underUVlightwhencomplexedwithDNA.Thegelis
photographed,andthedistancemigratedbyeachbandof
identicalDNAmoleculesismeasuredandcomparedwith
acalibrationcurvetodeterminethesizeofeachfragment.
5. AnexampleofrestrictionsitemappingisshowninFigure
16.14.Arealrestrictionmapismorecomplexto
generate,involvingmorerestrictionsites,andmoresites
foreachenzyme.
6. Restrictionmappingmaybedonewithacircularplasmid,
aclonedsequence,orafragmentofplasmidpreparedby
gelcutting.
7. ToconfirmthattheorientationofaclonedDNAinsertis
correct,restrictionenzymesareselectedthatwillgive
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Chapter7slide42
differentresultsinoppositeorientations(Figure8.15).

SouthernBlotAnalysisofSequencesinthe
Genome
1.Knowingthelocationofrestrictionsitesinagenomeregionofinterestmaybe
usefulforanalyzingintronorganizationorcloningpartsofageneintoavector.
2.TherestrictionsitemapcanbedeterminedusingcDNAorthesamegenefroma
relatedspeciesasprobe.Theprocessisasfollows(Figure8.16):
a.GenomicDNAsamplesarecutwithdifferentrestrictionenzymes.
b.Eachsampleiselectrophoresedinanagarosegel,andstainedwithethidium
bromide.GenomicDNAproducesalargevarietyoffragments,whichappearasa
smearonthegel.
c.DNAisdenaturedtosinglestrands,andSouthernblottingtransferstheDNAtoa
membranefilter.TheDNAfragmentsonthefilterarearrangedjustastheywerein
thegel.
d.Labeledprobeisaddedtothefilter,whereitwillhybridizewithanycomplementary
DNAfragmentsthatwereontheoriginalgel.BoundDNAisvisualizedas
appropriateforthelabeltype.
e.Thebandsshowingahybridizationsignalarecomparedwithmarkerbandsto
determinetheirsize,andconstructionofamapisbegun.Additionaldatafrom
individualandcombinedrestrictiondigestsmaybeneededtocompletethemap.
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Chapter7slide43

Fig. 8.16 Southern blot procedure

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

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Chapter7slide44

NorthernBlotAnalysisofRNA
1. NorthernblottinganalyzesRNAmuchthesamewaythatSouthern
blottingdoesDNA:
a. RNAisextractedfromthecell,undergoesgelelectrophoresisandis
boundtoafilter.
b. HybridizationbetweenboundcellularRNAandalabeledprobeoccurs.
ThesizesoftheRNAfragmentsdetectedbytheprobecanbe
determined.

2. Northernblotanalysisisusedfordetermining:
a. Thesize(s)ofmRNAencodedbyagene.Northernblotshaveshown
thatdifferentmRNAspeciesarisefromthesameregionofDNA,
suggestingdifferentialuseofpromotersandterminators,and/or
alternativemRNAprocessing.
b. WhetheraspecificmRNAispresentinacelltype,andifso,atwhat
levels.Geneactivityismeasuredinthisway,andRNAsamplingis
widelyusedtostudydevelopment,tissuespecialization,ortheresponse
ofcellstovariousphysiologicalstimuli.
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Chapter7slide45

DNASequencing
Animation:DideoxyDNASequencing
1. Oncecloned,DNAfragmentsmaybesequenced,
allowingidentificationofgeneandregulatory
sequences,andcomparisonwithhomologous
genesfromdifferentorganisms.

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Chapter7slide46

2.Dideoxysequencing(Sanger,1970s)isbasedonDNA
polymeraseextendingshortprimers,usingeitherlinearor
circularDNAasatemplate.Indideoxysequencing
(Figure8.17):
a.DNAisheatdenatured,andashortoligonucleotideprimer
(designedso3endisnearDNAsequenceofinterest)annealsto
onestrandandservesasprimer.
b.Areactionmixissetupwithlabeloneithertheprimeror
dNTP(s).Themixincludes:
i.ssDNAtemplatetobesequenced.
ii.Primer(annealstotemplate).
iii.DNApolymerase.
iv.Thefourdeoxynucleotide(dNTP)precursors.
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Chapter7slide47

Fig. 8.17 Dideoxy DNA sequencing of a theoretical DNA fragment

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Chapter7slide48

c.Thereactionmixisdividedintofourtubes,andtoeachisadded
asmallamountofadifferentdideoxynucleotide,sothatonetube
receivesddCTP,anotherddTTP,etc.(Figure8.18).
d.Dideoxynucleotideslacks3OH,havingonly3H,andsoare
unabletobondwiththe5phosphateofanothernucleotide.A
phosphodiesterlinkagecannotform,andthechainterminatesat
thesiteofinsertionoftheddNTP.
e.ThespecificddNTPinareactioncompeteswithits
correspondingNTPforincorporationintothegrowingDNA
chain.
f.Thefourreactionmixes,oneforeachddNTP,aretypicallyrunin
adjacentlanesonapolyacrylamidegel.ThelabelontheDNA
bandsrevealstheirlocation,andthereforetheirsizes.
g.DNAsequenceisdeterminedbyreadingthesequencingladder
frombottomtotoptogivethesequenceofthenewlysynthesized
strandfrom53(Figure8.19).
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Chapter7slide49

Fig. 8.18 A dideoxynucleotide (ddNTP) DNA precursor

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Chapter7slide50

Fig. 8.19 Autoradiogram of a dideoxy sequencing gel

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Chapter7slide51

3. Automationbasedonthedideoxymethodenables
rapidDNAsequencing.Intheautomatedprocess
(Figure8.20):
a. Onlyonereactionmixisneeded,containingallfour
dideoxynucleotides,eachtaggedwithadifferentcolor.
b. DNAfragmentsgeneratedareseparatedby
electrophoresisinasinglelane.
c. Thegelisscannedbyalaserdevicethatdetermines
whichfluorescentlabelispresentateachnucleotide
positioninthesequence,andsendstheinformationto
acomputer.
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Chapter7slide52

Fig. 8.20 Results of automated DNA sequence analysis using fluorescent dyes

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Chapter7slide53

4. ComputerprogramscananalyzeDNAsequencesusing
newdataalongwithdatabasesofpreviouslyreported
sequences.Computeranalysisisusedto:
a. Lookforrestrictionsites.
b. Compareavarietyofsequencesfromthesameordifferent
species.
c. Locatehomologousregions.
d. Findtranscriptionregulatorysequences.
e. Detectopenreadingframesandpredicttheaminoacidsequence
encoded,includingproteinstructureandfunction.
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Chapter7slide54

PolymeraseChainReaction(PCR)
1. PCRstartswithamixtureofDNAmoleculesandproducesmanycopiesofone
specificDNAsequence(amplimers).Mullisdevelopedthetechniqueinthe
1980s.
2. PCRbeginswithDNAcontainingthesequencetobeamplified,andapairof
syntheticprimersthatflankthesequence.Stepsintheprocessinclude(Figure
8.21):
a. HeatdenatureDNA(9495C).
b. Cool(3765C)andannealprimerstocomplementarysequenceswiththeir3ends
facingeachother,flankingthetargetDNA.
c. Extendtheprimers(7075C)withheatresistantDNApolymerasefromthe
thermophilicbacterium,Thermusaquaticus.
d. Repeatthecycleofdenaturationandprimerbinding.
e. ExtendagainwithTaqDNApolymerase.Productsthelengthofthetargetsequence
begintobeproduced.
f. Repeattheprocess,doublingtheamountoftargetDNAwitheachroundof
extension.

3. In20PCRcycles,millionfoldamplificationofthetargetsequenceoccurs.The
temperaturechangesareautomatedinathermalcycler.
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Fig. 8.21 The polymerase chain reaction (PCR) for selective amplification of DNA
sequences

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

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AdvantagesandLimitationsofPCR
1. PCRismoresensitiveandfasterthancloning,buttherearelimitations:
a. Specificprimersrequirethatsequenceinformationbeknown.
b. Taqpolymerasedoesnotproofread,meaningthatmismatchesgouncorrected.
AlternativepolymerasessuchasVentpolymerasedoproofread,decreasingerrors.
c. Thesensitivitycanresultinamplificationofcontaminatingsequences,aspecial
hazardinforensicapplications.

2. ApplicationsofPCRinclude:
a. AmplifyingDNAforcloning.
b. AmplifyingDNAfromgenomicDNAforsequencingwithoutcloning.
c. MappingDNAsegments.
d. Diseasediagnosis.
e. SubcloningsegmentsofclonedDNA(e.g.,theyeastARG1gene)
i.IndividualgenesmaybeamplifiedfromaclonedmultigeneDNAfragment.
ii.Complementationisusedtodeterminefunctionsofeachgene.
f. Forensics(theanalysisoflegalevidence)insamplesincludinghair,blood,orsemen.
g. Thestudyofmolecularevolution.
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RTPCRandmRNAQuantification
1. ReversetranscriptaseallowsamplificationofRNAbyPCRintwosteps:
a. cDNAissynthesizedfromRNAusingoligo(dT)asprimerandreversetranscriptase
aspolymerase.
b. ThecDNAisamplifiedbyPCR.

2. RTPCRcan:
a. TestforpresenceofanRNA(e.g.,detectRNAvirusgenomes).
b. QuantifyanmRNAtodeterminetheamountofgeneexpression.
i.DNAproductisanalyzedbyagarosegelelectrophoresiswithethidium
bromide.
ii.AmountofproductisvisualizedbyintensityoffluorescencewithUV.
iii.Resultsareonlysemiquantitative.Northernblotsaremoreaccuratefor
quantifyingmRNAs.

3. RealtimeRTPCRisalsomoreaccuratetoquantifymRNAlevels.
a. Reversetranscriptaseisusedforthefirststep,asinRTPCR.
b. DNAamplificationdoneinthepresenceofSYBRgreen,adyethatstainsdsDNA.
c. LaserdetectorinthethermalcyclerdetectsSYBRgreenfluorescence,quantifying
product.
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