【精品文档】QCT骨密度仪与其它骨密度检测技术的全方位比较相关交流-蚂蚁淘在线

【精品文档】QCT骨密度仪与其它骨密度检测技术的全方位比较

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MichaelP.Weiner,TimGackstetter,GinaL.Costa,JohnC.Bauer,andKeithA.Kretz

From:MolecularBIOLOGy:CurrentInnovationsandFutureTrends.Eds.A.M.GriffinandH.G.Griffin.ISBN1-898486-01-81995HorizonScientificPress,POBox1,Wymondham,Norfolk,U.K.

Invitrosite-directedmutagenesisisaninvaluabletechniqueforstudyingproteinstructure-functionrelationships,geneexpressionandvectormodification.Severalmethodshaveappearedintheliterature,butmanyofthesemethodsrequiresingle-strandedDNAasthetemplate.Thereasonforthis,historically,hasbeentheneedforseparatingthecomplementarystrandstopreventreannealing.UseofPCRinsite-directedmutagenesisaccomplishesstrandseparationbyusingadenaturingsteptoseparatethecomplementingstrandsandallowingefficientpolymerizationofthePCRprimers.PCRsite-directedmethodsthusallowsite-specificmutationstobeincorporatedinvirtuallyanydouble-strandedplasmid;eliminatingtheneedforM13-basedvectorsorsingle-strandedrescue.

Severalpointsshouldbementionedconcerningsite-directedmutagenesisusingPCR.First,itisoftendesirabletoreducethenumberofcyclesduringPCRwhenperformingPCR-basedsite-directedmutagenesistopreventclonalexpansionofany(undesired)second-sitemutations.Limitedcyclingwhichwouldresultinreducedproductyield,isoffsetbyincreasingthestartingtemplateconcentration.Second,aselectionmustbeusedtoreducethenumberofparentalmoleculescomingthroughthereaction.Third,inordertouseasinglePCRprimerset,itisdesirabletooptimizethelongPCRmethod.Andfourth,becauseoftheextendaseactivityofsomeThermostablepolymerasesitisoftennecessarytoincorporateanend-polishingstepintotheprocedurepriortoend-to-endligationofthePCR-generatedproductcontainingtheincorporatedmutationsinoneorbothPCRprimers.

Aprotocolisprovidedasafacilemethodforsite-directedmutagenesisandaccomplishestheabovedesiredfeaturesbytheincorporationofthefollowingsteps:(i)increasingtemplateconcentrationapproximately1000-foldoverconventionalPCRconditions;(ii)reducingthenumberofcyclesfrom25-30to5-10;(iii)addingtherestrictionendonucleaseDpnI(recognitiontargetsequence:5-Gm6ATC-3,wheretheAresidueismethylated)toselectagainstparentalDNA(note:DNAisolatedfromalmostallcommonstrainsofE.coliisDam-methylatedatthesequence5-GATC-3);(iv)usingTaqExtenderinthePCRmixforincreasedreliABIlityforPCRto10kb;(v)usingPfuDNApolymerasetopolishtheendsofthePCRproduct,and(vi)efficientintramolecularligationinthepresenceofT4DNAligase.

Protocol.PCR-basedSiteDirectedMutagenesis

PlasmidtemplateDNA(approximately0.5pmole)isaddedtoaPCRcocktailcontaining,in25ulof1xmutagenesisbuffer:(20mMTrisHCl,pH7.5;8mMMgCl2;40ug/mlBSA);12-20pmoleofeachprimer(oneofwhichmustcontaina5-primephosphate),250uMeachdNTP,2.5UTaqDNApolymerase,2.5UofTaqExtender(Stratagene).
ThePCRcyclingparametersare1cycleof:4minat94C,2minat50Cand2minat72C;followedby5-10cyclesof1minat94C,2minat54Cand1minat72C(step1).
TheparentaltemplateDNAandthelinear,mutagenesis-primerincorporatingnewlysynthesizedDNAaretreatedwithDpnI(10U)andPfuDNApolymerase(2.5U).ThisresultsintheDpnIdigestionoftheinvivomethylatedparentaltemplateandhybridDNAandtheremoval,byPfuDNApolymerase,oftheTaqDNApolymerase-extendedbase(s)onthelinearPCRproduct.
Thereactionisincubatedat37Cfor30minandthentransferredto72Cforanadditional30min(step2).
Mutagenesisbuffer(1x,115ul,containing0.5mMATP)isaddedtotheDpnI-digested,PfuDNApolymerase-polishedPCRproducts.
Thesolutionismixedand10ulisremovedtoanewmicrofugetubeandT4DNAligase(2-4U)added.
Theligationisincubatedforgreaterthan60minat37C(step3).
ThetreatedsolutionistransformedintocompetentE.coli(step4).

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