Preparation of nucleic acid probes

Preparationofnucleicacidprobes

Instandardnucleicacidhybridizationassaystheprobeislabeledinsomeway.Nucleicacidprobesmaybemadeassingle-strandedordouble-strandedmolecules(seeFigure5.1),buttheworkingprobemustbeintheformofsinglestrands.

ConventionalDNAprobesareisolatedbycell-basedDNAcloningorbyPCR.Intheformercase,thestartingDNAmayrangeinsizefrom0.1kbtohundredsofkilobasesinlengthandisusually(butnotalways)originallydouble-stranded.PCR-derivedDNAprobeshaveoftenbeenlessthan10kblongandareusually,butnotalways,originallydouble-stranded.ConventionalDNAprobesareusuallylabeledbyincorporatinglabeleddNTPsduringaninvitroDNAsynthesisreaction(seeSection5.1.1).

RNAprobescanconvenientlybegeneratedfromDNAwhichhasbeenclonedinaspecializedplasmidvector(Meltonetal.,1984).Suchvectorsnormallycontainaphagepromotersequenceimmediatelyadjacenttothemultiplecloningsite.AnRNAsynthesisreactionisemployedusingtherelevantphageRNApolymeraseandthefourrNTPs,atleastoneofwhichislabeled.SpecificlabeledRNAtranscriptscanthenbegeneratedfromtheclonedinsert(seeSection5.1.1).

Oligonucleotideprobesareshort(typically1550nucleotides)single-strandedpiecesofDNAmadebychemicalsynthesis:mononucleotidesareadded,oneatatime,toastartingmononucleotide,conventionallythe3endnucleotide,whichisboundtoasolidsupport.Generally,oligonucleotideprobesaredesignedwithaspecificsequencechoseninresponsetopriorinformationaboutthetargetDNA.Sometimes,however,oligonucleotideprobesareusedwhicharedegenerateinsequence.Typicallythisinvolvesparallelsynthesesofasetofoligonucleotideswhichareidenticalatcertainnucleotidepositionsbutdifferentatothers.Oligonucleotideprobesareoftenlabeledbyincorporatinga³²Patomorotherlabeledgroupatthe5end(seenextsection).

Although,inprinciple,DNAandRNAcanbelabeledinvivo,bysupplyinglabeleddeoxynucleotidestotissueculturecells,thisprocedureisoflimitedgeneraluse;ithasbeenrestrictedlargelytopreparinglabeledviralDNAfromvirus-infectedcells,andstudyingRNAprocessingevents.Amuchmoreversatilemethodinvolvesinvitrolabeling:thepurifiedDNA,RNAoroligonucleotideislabeledinvitrobyusingasuitableenzymetoincorporatelabelednucleotides.Twomajortypesofprocedurehavebeenwidelyused:

• LabelingofnewstrandsduringinvitroDNAorRNAsynthesis.Inthistypeofprocedure,DNAorRNApolymeraseisusedtomakelabeledDNAorRNAcopiesofastartingDNA.TheinvitroDNAorRNAsynthesisreactionrequiresthatatleastoneofthefournucleotideprecursorscarriesalabeledgroup.LabelingofDNAbyinvitroDNAsynthesisisnormallyaccomplishedusingoneofthreemethods:nick-translation(thissection);randomprimedlabeling(thissection);orPCR-mediatedlabeling(seeSection6.1.1).LabelingofRNAgenerallyiscarriedoutusinganinvitrotranscriptionsystem(seefurtheroninthissection).
• End-labeling.Thistypeofprocedureinvolvesadditionofalabeledgrouptooneorafewterminalnucleotides.Itislesswidelyused,butisusefulforanumberofprocedures,includinglabelingofsingle-strandedoligonucleotides(seebelow)andrestrictionmapping.Inevitably,becauseonlyoneoraveryfewlabeledgroupsareincorporated,thespecificactivity(theamountofrADIoactivityincorporateddividedbythetotalmass)ofthelabeledDNAismuchlessthanthatforprobesinwhichtherehasbeenincorporationofseverallabelednucleotidesalongthelengthoftheDNA.

Thenick-translationprocedureinvolvesintroducingsingle-strandbreaks(nicks)intheDNA,leavingexposed3hydroxylterminiand5phosphatetermini.ThenickingcanbeachievedbyaddingasuitableendonucleasesuchaspancreaticdeoxyribonucleaseI(DNaseI).Theexposednickcanthenserveasastartpointforintroducingnewnucleotidesatthe3hydroxylsideofthenickusingtheDNApolymeraseactivityofE.coliDNApolymeraseIatthesametimeasexistingnucleotidesareremovedfromtheothersideofthenickbythe53exonucleaseactivityofthesameenzyme.Asaresult,thenickwillbemovedprogressivelyalongtheDNA("translated")inthe53direction(seeFigure5.2A).Ifthereactioniscarriedoutatarelativelylowtemperature(about15°C),thereactionproceedsnofurtherthanonecompleterenewaloftheexistingnucleotidesequence.AlthoughthereisnonetDNAsynthesisatthesetemperatures,thesynthesisreactionallowstheincorporationoflabelednucleotidesinplaceofthepreviouslyexistingunlabeledones.

TherandomprimedDNAlabelingmethod(sometimesknownasoligolabeling)(FeinbergandVogelstein,1983)isbasedonhybridizationofamixtureofallpossIBLehexanucleotides:thestartingDNAisdenaturedandthencooledslowlysothattheindividualhexanucleotidescanbindtosuitablycomplementarysequenceswithintheDNAstrands.SynthesisofnewcomplementaryDNAstrandsisprimedbytheboundhexanucleotidesandiscatalyzedbytheKlenowsubunitofDNApolymeraseI(whichcontainsthepolymeraseactivityintheabsenceofassociatedexonucleaseactivities).DNAsynthesisoccursinthepresenceofthefourdNTPs,atleastoneofwhichhasalabeledgroup(seeFigure5.2B).ThismethodproduceslabeledDNAsofhighspecificactivity.Becauseallsequencecombinationsarerepresentedinthehexanucleotidemixture,bindingofprimertotemplateDNAoccursinarandommanner,andlabelingisuniformacrossthelengthoftheDNA.

Single-strandedoligonucleotidesareusuallyend-labeledusingpolynucleotidekinase(kinaseend-labeling).Typically,thelabelisprovidedintheformofa³²Pattheg-phosphatepositionofATPandthepolynucleotidekinasecatalysesanexchangereactionwiththe5-terminalphosphates(seeFigure5.3A).Thesameprocedurecanalsobeusedforlabelingdouble-strandedDNA.Inthiscase,fragmentscarryinglabelatoneendonlycanthenbegeneratedbycleavageataninternalrestrictionsite,generatingtwodifferentlysizedfragmentswhichcanbeseparatedbygelelectrophoresisandpurified.

LargerDNAfragmentscanbeend-labeledbyvariousalternativemethods.Fill-inend-labeling(Figure5.3B)isonepopularapproach,andusestheKlenowsubunitofE.coliDNApolymerase.Again,fragmentscarryinglabelatoneendonlycanbegeneratedbyrestrictioncleavageandsizefractionation.AnalternativePCR-basedmethodisprimer-mediated5end-labeling(seeSection6.1.1).

ThepreparationoflabeledRNAprobes(riboprobes)ismosteasilyachievedbyinvitrotranscriptionofinsertDNAclonedinasuitableplasmidvector.Thevectorisdesignedsothatadjacenttothemultiplecloningsiteisaphagepromotersequence,whichcanberecognizedbythecorrespondingphageRNApolymerase.Forexample,theplasmidvectorpSP64containsthebacteriophageSP6promotersequenceimmediatelyadjacenttoamultiplecloningsite(seeFigure5.4).TheSP6RNApolymerasecanthenbeusedtoinitiatetranscriptionfromaspecificstartpointintheSP6promotersequence,transcribingthroughanyDNAsequencethathasbeeninsertedintothemultiplecloningsite.ByusingamixofNTPs,atleastoneofwhichislabeled,highspecificactivityradiolabeledtranscriptscanbegenerated(Figure5.4).BacteriophageT3andT7promoter/RNApolymerasesystemsarealsousedcommonlyforgeneratingriboprobes.Labeledsenseandantisenseriboprobescanbegeneratedfromanygeneclonedinsuchvectors(thegenecanbeclonedineitherofthetwoorientations)andarewidelyusedintissueinsituhybridization(Section5.3.4).

Traditionally,labelingofnucleicacidshasbeenconductedbyincorporatingnucleotidescontainingradioisotopes.Suchradiolabeledprobescontainnucleotideswitharadioisotope(often³²P,³³P,³⁵Sor³H),whichcanbedetectedspecificallyinsolutionor,muchmorecommonly,withinasolidspecimen(autoradiography-seeBox5.1).

Theintensityofanautoradiographicsignalisdependentontheintensityoftheradiationemittedbytheradioisotope,andthetimeofexposure,whichmayoftenbelong(oneormoredays,orevenweeksinsomeapplications).³²PhasbeenusedwidelyinSouthernblothybridization,dot-blothybridization,colonyandplaquehybridization(seebelow)becauseitemitshighenergyb-particleswhichaffordahighdegreeofsensitivityofdetection.Ithasthedisadvantage,however,thatitisrelativelyunstable(seeTable5.1).Additionally,itshighenergyb-particleemissioncanbeadisadvantageundercircumstanceswhenfinephysicalresolutionisrequiredtointerprettheresultingimageunambiguously.Forthisreason,radionuclideswhichprovidelessenergeticb-particleradiationhavebeenpreferredincertainprocedures,forexample³⁵S-labeledand³³P-labelednucleotidesforDNAsequencingandtissueinsituhybridization,and³H-labelednucleotidesforchromosomeinsituhybridization.³⁵Sand³³Phavemoderatehalf-liveswhile³Hhasaverylonghalf-life.However,thelatterisotopeisdisadvantagedbyitscomparativelylowenergyb-particleemissionwhichnecessitatesverylongexposuretimes.

³²P-labeledand³³P-labelednucleotidesusedinDNAstrandsynthesislabelingreactionshavetheradioisotopeatthea-phosphateposition,becausetheb-andg-phosphatesfromdNTPprecursorsarenotincorporatedintothegrowingDNAchain.Kinase-mediatedend-labeling,however,uses[g-³²P]ATP(seeFigure5.3A).Inthecaseof³⁵S-labelednucleotideswhichareincorporatedduringthesynthesisofDNAorRNAstrands,theNTPordNTPcarriesa³⁵SisotopeinplaceoftheO^-ofthea-phosphategroup.³H-labelednucleotidescarrytheradioisotopeatseveralpositions.Specificdetectionofmoleculescarryingaradioisotopeismostoftenperformedbyautoradiography(seeBox5.1).

Nonisotopiclabelingsystemsinvolvetheuseofnonradioactiveprobes.Althoughdevelopedonlycomparativelyrecently,theyarebecomingincreasinglypopularandarefindingincreasingapplicationsinavarietyofdifferentareas(Kricka,1992).Twotypesofnon-radioactivelabelingareconducted:

• Directnonisotopiclabeling,whereanucleotidewhichcontainsthelabelthatwillbedetectedisincorporated.Oftensuchsystemsinvolveincorporationofmodifiednucleotidescontainingafluorophore(Figure5.5A),achemicalgroupwhichcanfluorescewhenexposedtolightofacertainwavelength(fluorescencelabeling-seeBox5.2).
• Indirectnonisotopiclabeling,usuallyfeaturingthechemicalcouplingofamodifiedreportermoleculetoanucleotideprecursor.AfterincorporationintoDNA,thereportergroupscanbespecificallyboundbyanaffinitymolecule,aproteinorotherligandwhichhasaveryhighaffinityforthereportergroup.ConjugatedtothelatterisaMarkermoleculeorgroupwhichcanbedetectedinasuitableassay(Figure5.6).Thereportermoleculesonmodifiednucleotidesneedtoprotrudesufficientlyfarfromthenucleicacidbackbonetofacilitatetheirdetectionbytheaffinitymoleculeandsoalongcarbonatomspacerisrequiredtoseparatethenucleotidefromthereportergroup.

Twoindirectnonisotopiclabelingsystemsarewidelyused:

• Thebiotin-streptavidinsystemutilizestheextremelyhighaffinityoftwoligands:biotin(anaturallyoccurringvitamin)whichactsasthereporter,andthebacterialproteinstreptavidin,whichistheaffinitymolecule.Biotinandstreptavidinbindtogetherextemelytightlywithanaffinityconstantof10^-14,oneofthestrongestknowninBIOLOGy.Biotinylatedprobescanbemadeeasilybyincludingasuitablebiotinylatednucleotideinthelabelingreaction(seeFigure5.7).
• Digoxigeninisaplantsteroid(obtainedfromDigi-talisplants)towhichaspecificantibodyhasbeenraised.Thedigoxigenin-specificantibodypermitsdetectionofnucleicacidmoleculeswhichhaveincorporatednucleotidescontainingthedigoxigeninreportergroup(seeFigure5.7).

Avarietyofdifferentmarkergroupsormoleculescanbeconjugatedtoaffinitymoleculessuchasstreptavidinorthedigoxigenin-specificantibody.Theyincludevariousfluorophores(seeBox5.2),orenzymessuchasalkalinephosphataseandperoxidasewhichcanpermitdetectionviacolorimetricassaysorchemicalluminescenceassays,etc.

Figure5.1.Originandcharacteristicsofnucleicacidhybridizationprobes.

Figure5.2.DNAlabelingbyinvitroDNAstrandsynthesis.(A)Nicktranslation.PancreaticDNaseIintroducessingle-strandednicksbycleavinginternalphosphodiesterbonds(p),generatinga5phosphategroupanda3hydroxylterminus.AdditionofthemultisubunitenzymeE.coliDNApolymeraseIcontributestwoenzymeactivities:(i)a53exonucleaseattackstheexposed5terminiofanickandsequentiallyremovesnucleotidesinthe53direction;(ii)aDNApolymeraseaddsnewnucleotidestotheexposed3hydroxylgroup,continuinginthe53direction,therebyreplacingnucleotidesremovedbytheexonucleaseandcausinglateraldisplacement(translation)ofthenick.(B)Randomprimedlabeling.TheKlenowsubunitofE.coliDNApolymeraseIcansynthesizenewradiolabeledDNAstrandsusingasatemplateseparatedstrandsofDNA,andrandomhexanucleotideprimers.

Figure5.3.End-labelingofDNA.(A)Kinaseend-labelingofoligonucleotides.The5-terminalphosphateoftheoligonucleotideisreplacedinanexchangereactionbythe³²P-labeledg-phosphateof[g-³²P]ATP.Thesameprocedurecanbeusedtolabelthetwo5terminiofdouble-strandedDNA.(B)Fill-inend-labelingbyKlenow.TheDNAofinterestiscleavedwithasuitablerestrictionnucleasetogenerate5overhangs.TheoverhangsactasaprimerforKlenowDNApolymerasetoincorporatelabelednucleotidescomplementarytotheoverhang.Fragmentslabeledatoneendonlycanbegeneratedbyinternalcleavagewithasuitablerestrictionsitetogeneratetwodifferentlysizedfragmentswhichcaneasilybesize-fractionated.

Figure5.7.Structureofdigoxigenin-andbiotin-modifiednucleotides.Notethatthedigoxigeninandbiotingroupsintheseexamplesarelinkedtothe5carbonatomoftheuridineofdUTPbyspacergroupsconsistingrespectivelyofatotalof11carbonatoms(digoxigenin-11-UTP)or16carbonatoms(biotin-16-dUTP).Thedigoxigeninandbiotingroupsarereportergroups:afterincorporationintoanucleicacidtheyareboundbyspecificligandscontaininganattachedmarkersuchasafluorophore.