Академический Документы
Профессиональный Документы
Культура Документы
Russell
AmolecularApproach2ndEdition
CHAPTER 8
Recombinant DNA
Technology
editedbyYueWenWangPh.D.
Dept.ofAgronomy,NTU
60120000
Chapter7slide1
DNACloning
1. Thegoalofmolecularcloningislargeamountsofpure
DNAthatcanbefurthermanipulatedandstudied.
2. Summaryoftheprocedure:
a. IsolateDNAfromtheorganism.
b. UserestrictionenzymestocuttheDNA,andligatefragments
intoacloningvector.
c. TransformrecombinantDNAintoahost,whichwillreplicate
theDNA(molecularcloning)andpasscopiestoallprogeny.
60120000
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).
60120000
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.
60120000
Chapter7slide4
60120000
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).
60120000
Chapter7slide6
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
60120000
Chapter7slide7
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
60120000
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).
60120000
Chapter7slide9
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
60120000
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.
60120000
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.
60120000
Chapter7slide12
ShuttleVectors
1. Acloningvectorcapableofreplicatingintwoor
moretypesoforganism(e.g.,E.coliandyeast)is
calledashuttlevector.Shuttlevectorsmay
replicateautonomouslyinbothhosts,orintegrate
intothehostgenome
60120000
Chapter7slide13
ExpressionVectors
1. Expressionvectorshavesequencestoallowtranscription
andtranslationoftheclonedgene(s).Pharmaceuticals
producedbybiotechnologyareanexample.
2. Plasmidcloningvectorsaremodifiedtoinclude:
a. Promoterandtranscriptionterminatorifneeded,suitabletothe
hostorganism.
b. Anymodificationsneededtocrossprokaryotic/eukaryotic
boundary(e.g.,ShineDelgarnosequenceisaddedfortranslation
ofeukaryoticsequencesinE.coli).
60120000
Chapter7slide14
ArtificialChromosomes
1. Cloningvectorsthatcanaccommodateverylarge
piecesofDNAproducemoleculesresembling
smallchromosomes.TwoexamplesareYACs
andBACs.
60120000
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.
60120000
Chapter7slide16
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
60120000
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.
60120000
Chapter7slide18
iActivity:BuildingABetterBeer?
60120000
Chapter7slide19
RecombinantDNALibraries
1. Itissometimesusefultohaveagenomiclibrary,a
collectionofclonescontainingatleastonecopy
ofeveryDNAsequenceinagenome.Genomic
librariesareavailableformanyorganisms.
2. Chromosomelibrariesarecollectionsofcloned
fragmentsofindividualchromosomes.
3. ComplementaryDNA(cDNA)librariesare
clonedcollectionsofDNAcopiedfromacells
mRNA.
60120000
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.
60120000
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.
60120000
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.
60120000
Chapter7slide23
Chromosomelibraries
1. Screeningthegenomiclibraryofanorganism
withalargegenomeislaborious.Screeningtime
canbereducedifagenehasbeenlocalizedtoa
chromosome,byexaminingalibrarymadefrom
onlythatchromosome.Human,forexample,
have24differentchromosomelibraries(22
autosomes,XandY).
2. Separatingchromosomesotheymaybe
individuallyclonedisaccomplishedwith
techniquessuchasflowcytometry.
60120000
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.
60120000
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.
60120000
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.
60120000
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.
60120000
Chapter7slide28
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
60120000
Chapter7slide29
FindingaSpecificCloneinaLibrary
1. Anumberoftechniqueshavebeendevelopedfor
identifyingthecloneofinterestinacDNA
library.Somearediscussedhere.
60120000
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.
60120000
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.
60120000
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.
60120000
Chapter7slide33
Fig. 8.11 Using DNA probes to screen plasmid or cosmid genomic libraries for specific
DNA sequences
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
60120000
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.
60120000
Chapter7slide35
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
60120000
Chapter7slide36
IdentifyingSpecificDNASequencesinLibraries
UsingHeterologousProbes
60120000
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.
60120000
Chapter7slide38
MolecularAnalysisofClonedDNA
1. ClonedDNAisusedinmanytypesof
experiments.Threeexamplesare:
a. Restrictionmapping
b. Southernblotting
c. Northernblotting
60120000
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.
60120000
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.
60120000
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
60120000
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.
60120000
Chapter7slide43
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
60120000
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.
60120000
Chapter7slide45
DNASequencing
Animation:DideoxyDNASequencing
1. Oncecloned,DNAfragmentsmaybesequenced,
allowingidentificationofgeneandregulatory
sequences,andcomparisonwithhomologous
genesfromdifferentorganisms.
60120000
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.
60120000
Chapter7slide47
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
60120000
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).
60120000
Chapter7slide49
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
60120000
Chapter7slide50
60120000
Chapter7slide51
3. Automationbasedonthedideoxymethodenables
rapidDNAsequencing.Intheautomatedprocess
(Figure8.20):
a. Onlyonereactionmixisneeded,containingallfour
dideoxynucleotides,eachtaggedwithadifferentcolor.
b. DNAfragmentsgeneratedareseparatedby
electrophoresisinasinglelane.
c. Thegelisscannedbyalaserdevicethatdetermines
whichfluorescentlabelispresentateachnucleotide
positioninthesequence,andsendstheinformationto
acomputer.
60120000
Chapter7slide52
Fig. 8.20 Results of automated DNA sequence analysis using fluorescent dyes
60120000
Chapter7slide53
4. ComputerprogramscananalyzeDNAsequencesusing
newdataalongwithdatabasesofpreviouslyreported
sequences.Computeranalysisisusedto:
a. Lookforrestrictionsites.
b. Compareavarietyofsequencesfromthesameordifferent
species.
c. Locatehomologousregions.
d. Findtranscriptionregulatorysequences.
e. Detectopenreadingframesandpredicttheaminoacidsequence
encoded,includingproteinstructureandfunction.
60120000
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.
60120000
Chapter7slide55
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.
60120000
Chapter7slide56
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.
60120000
Chapter7slide57
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.
60120000
Chapter7slide58