DNAWorks全基因合成介绍课件

GeneSynthesisusingDNAWorksDr.DavidHooverHelixSystems,SCB,CIT,NIHGeneSynthesisSeveralmethodsligation-incrediblytediousandinefficientFokI-sequencedependent(typeIIsr.e.)serialcloning-sequencedependentassemblyorself-primingPCRGeneSynthesisMethodsThermodynamicallyBalancedConventionalThermodynamicallyBalancedInside-OutCodonBiasSyntheticGenesBenefits:CodonuseoptimizedforhostFlexibilityinsubcloningEaseofcomplexmutagenesisProblems:TimeconsumingComplicatedError-proneCommercialSourcesBlueHeronBiotechnology()DNA2.0()GeneScriptCorporation()BioNexusInc.()Entelechon()GeneArt()CodonDevices()CommericalSourcesTypicalcosts:$0.79-$3.60/bpComplexities?Intellectualproperty?800bp=$1000(GeneScript)Howtodesignoligosreverse-translateproteinintoDNA,optimumcodonusagebreakintofragmentsofequaloverlapTmoptimize:hairpins/mRNAstructurerepeats/misprimingrestrictionsiteinclusion/exclusionlengthDNAWorksOutput181TCTGGTGAAGGCGAGGGTGACGCGACCTACGGTAAACTCACTCTCAAATagaccactTGCCATTTGAGTGAGAGTTTAAGTAGACGTGG<---4SGEGEGDATYGKLTLKFICT|||||||7--->241ggttccttggccgaccctggttactaccttctcttacggtgttcagTGCCCGTTTGACGGCCAAGGAACCGGCTGGtc<---6TGKLPVPWPTLVTTFSYGVQ|||||||DNAWorksOptionsJobNameE-mailAddressDNAWorksOptionsCodonFrequencyTableE.coli(standard,classII),H.sapiens,C.elegans,D.melanogaster,M.musculus,P.pastoris,R.norvegicus,S.cerevesiae,X.laevisCustomCFTDNAWorksOptionsParametersAnnealingTemperatureOligoLength(random)CodonFrequencyThreshold(random,strict,scored)Oligonucleotide,Na+/K+,Mg2+ConcentrationsNumberofSolutionsTBIONogapsinassemblyDNAWorksOptionsBalancingactFast,simple,cheap?Slow,complex,expensive?-reliableReusableandinterchangeableoligos?DNAWorksOptionsOthersRestrictionSiteScreen(non-degenerate,degeneratesequences)CustomSiteScreen(mindtheformat!)Weights(experimental)DNAWorksOutputWeboutputInputforDNAWorks(standaloneversion)HeaderInitialparametersOptimizationlogFinalscoresFinalsummaryDNAWorksOutputTotaloutputSequenceblocksCFTblocksPatternblockTrialsFinalSummaryDNAWorksOutputTrialoutputsInitialparametersFinalDNAsequenceAssemblyFinalscoresCodonreportHistogramsOligosequencesMutantRunDesignoligosbasedonprevioussetofoligosParameterstakenfrompreviousrunForsinglemutation,willoutput1or2oligosonlyWhattolookforFinalSummaryAvoidmisprimesandrepeatsMakesureoverlapsare>12nt(Short)Tmrangeshouldnotbe>3°C(TmRange)Don'tdependentirelyonscoresArbitrary,somewhatdependentonlengthTricksThermodynamicallyBalancedInside-OutModeMulti-stepPCRMorecontrolled,reliablemethodGaoX.,etal.,NucleicAcidsRes2003RandomoligolengthsFaster,betteroptimizationForthenot-so-fussyProbablybestforDNA-onlygenesTricksSetTmhigher64°C-70°Clongeroligos,extrapurification($$$)Alwaysdoublecheck!NothingisfoolproofThinkcarefullyaboutwhatyouneedBEFOREstartingworkAlwaysrunfinalsequencesthroughalternateprogram(EMBOSS,GCG-Lite)MakesureoligosarewhatyouintendedPCRMixalloligosandadditivesSpecificPCRprotocolsAnalyticalgelIsolatedesiredproductsAssemblyProtocolOligos1μl625nMeach25nMeachdNTPs2μl2.5mMeach0.25mMeachH2O19μlBuffer2.5μl10X1XPfupol.0.5μl95°C2.0'1X95°C0.5'65>55°(-0.5)0.5'20X72°C0.5'72°C5'1X4°CholdAmplificationProtocolPCRmix2μl??dNTPs8μl2.5mMeach0.2mMeach3'primer4μl10μM400nM5'primer4μl10μM400nMBuffer10μl10X1XH2O70μlPfupol.2μl95°C2.0'1X95°C0.5'62°C0.5'20X72°C0.5'72°C5'1X4°CholdProblemsNoproduct(completefailure)Wrongsizeproduct(mispriming)Mutations(2outof3correct,2errors/kb)Sequencingiswarranted...FixesOptimizePCRconditionsBreakgenesynthesisintosteps(TBIO)Errorsp=mutationrate/1000nt/duplication(Clineetal.,NucleicAcidsRes24(1996))Taqpolymerase=0.008KOD(Novagen)=0.0027PfuUltra(Stratagene)=0.00043Theprobabilityofagenenbpinlengthhavingnoerrorsusingapolymerasewithmutationratep: p'=(1-p)nTherefore,p'fora738bpgene=(1-0.00043)738=0.728ErrorsThenumberofclonesneededtoscreentofindacorrectgenewith95%confidence: N=log(0.05)/log(1-p')Thus,log(0.05)/log(1-0.728)=3clonesneedtobesequenced.FromWuetal.,JBiotech124(2006)TimeFindproteinofinterest,designoligos,orderoligosRunPCR,integrateintosequencingvector,transformPick