Robust one-Tube (cid:2)-PCR Strategy Accelerates Precise Sequence Modification of Plasmids for Functional Genomics Letian Chen1,2,3,*, Fengpin Wang1,2,3, Xiaoyu Wang1,2 and Yao-Guang Liu1 1StateKeyLaboratoryforConservationandUtilizationofSubtropicalAgro-bioresources,CollegeofLifeSciences,SouthChina AgriculturalUniversity,Guangzhou510642,China 2GuangdongProvincialKeyLaboratoryofProteinFunctionandRegulationinAgriculturalOrganisms,CollegeofLifeSciences,South ChinaAgriculturalUniversity,Guangzhou510642,China T 3Theseauthorscontributedequallytothiswork. e *Correspondingauthor:E-mail,[email protected];Fax,+86-20-85282180. c h (Received October 24, 2012; Accepted January 10, 2013) n i q Functional genomics requires vector construction for pro- suitable restriction site(s) in a specific vector and/or a target u tein expression and functional characterization of target sequencecanbeproblematic.Theseobstaclescanbepartially e s genes; therefore, a simple, flexible and low-cost molecular overcome by in vitro site-specific recombinational cloning manipulation strategy will be highly advantageous for gen- (Hartley et al. 2000) and by In-FusionTM assembly (Zhu et al. omicsapproaches.Here,wedescribe a(cid:2)-PCRstrategythat 2007), because of their fast reaction speeds and restriction enables multiple types of sequence modification, including enzyme-freenature.Althoughthesetechnologiesarecommer- precise insertion, deletion and substitution, in any position cially available as Invitrogen TOPO Gateway system and of a circular plasmid. (cid:2)-PCR is based on an overlap exten- Clontech In-Fusion(cid:2) kits, the high cost of the kits restricts sion site-directed mutagenesis technique, and is named for theirutilityforroutinevectorconstructioninmostmolecular itscharacteristic(cid:2)-shapedsecondarystructureduringPCR. biology labs. Alternatively, numerous PCR-based cloning stra- (cid:2)-PCRcanbeperformedeitherintwosteps,orinonetube tegies and site-directed mutagenesis methods have been de- incombination withexonucleaseItreatment. Thesestrate- veloped for their technical simplicity, low cost and high gies have wide applications for protein engineering, gene efficiency (Kunkel 1985, Kammann et al. 1989, Marchuk et al. functionanalysis andinvitro gene splicing. 1991, Datta 1995, Ke and Madison 1997, Bryksin and Matsumura 2010), but most of these methods preferentially Keywords: Gene cloning (cid:2) In vitro gene splicing (cid:2) Molecular allowinsertionalsequencemodification. manipulation (cid:2) (cid:2)-PCR. In1989,Hoetal.reporteddevelopment oftheoverlap ex- tensionsite-directedmutagenesistechnique,whichenabledall Abbreviations: Den, Dendra2 fluorescent protein; gDNA, threesequencemodifications(Hoetal.1989)andwasadopted genomic DNA; GFP, green fluorescent protein; (cid:2)-PCR, intheStratageneQuikChangeTMSite-DirectedMutagenesisKit. omega-PCR; TAC, transformation-competent artificial This method requires supercoiled double-stranded DNA plas- chromosome. mid as template, two synthetic complementary oligonucleo- tides containing the desired point mutations as primers and Introduction themethylation-specificendonucleaseDpnItoremovethepar- entalDNAtemplate.Kammannetal.(1989)firstreportedthe Inthepost-genomicera,examinationofproteinpropertiesand useofdouble-strandedPCRproductsasPCRprimers.Asimilar functions of specific genes in transient and stable systems concept,using‘megaprimers’,wasdevelopedandmodifiedina requires specific, nucleotide-level modification of large num- number of PCR-based mutagenesis protocols to introduce a bers of vector constructs carrying target genes. Therefore, a mutation into a gene of interest (Giebel and Spritz 1990, simple,flexible,low-costandhigh-fidelitymethodforsequence Sarkar and Sommer 1990, Datta 1995, Ke and Madison 1997, modification is highly desirable for these processes. Sequence Tyagi et al. 2004). However, these protocols incorporate the modification requires three types of manipulation: insertion, modification, particularly insertions orsubstitutions, into syn- substitutionanddeletion.Inconventionalstrategies,thesese- theticprimers,whichlimitsthecapacityofPCR-basedmodifi- quencemodificationsareachievedinacut-and-pastemanner cation of longer sequences. To by-pass the primer length basedonrestrictionendonucleasesandmodificationenzymes limitation,hereweintroduceaspecialdesignforchimericpri- suchasligases,phosphatases,kinasesandothers.Thisistedious, mersandincorporateuseofPCRproductsasmegaprimersinto time consuming and therefore expensive. In addition, finding the site-directed mutagenesis protocol, establishing a set of PlantCellPhysiol.54(4):634–642 (2013) doi:10.1093/pcp/pct009,availableonlineatwww.pcp.oxfordjournals.org !TheAuthor2013.PublishedbyOxfordUniversityPressonbehalfofJapaneseSocietyofPlantPhysiologists. ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense (http://creativecommons.org/licenses/by-nc/3.0/),whichpermitsunrestrictednon-commercialuse,distribution,andreproduction inanymedium,providedtheoriginalworkisproperlycited. 634 PlantCellPhysiol.54(4):634–642 (2013) doi:10.1093/pcp/pct009 !TheAuthor2013. (cid:2)-PCRforprecisesequencemodification simple,effectiveandhighcapacityPCR-basedmolecularengin- plasmids isolated from Escherichia coli are usually methylated eeringstrategies.ThemegaprimersgeneratedfromregularPCR and can be digested by the restriction endonuclease withhigh-fidelityDNApolymerasearecomplementary,blunt- DpnI, whereas in vitro synthesized DNA (PCR products) is ended and suitable for overlap extension site-directed muta- resistanttothisenzyme(WeinerandCosta1994).Aftertreat- genesis.Therefore,thecapacityforsequencemodificationisno mentwithDpnItoremovetheoriginaltemplateplasmids,the longerlimitedbyprimersynthesis.Thesestrategiesmakeuseof PCR product was transferred into E. coli. The pBI-Den:OsRac3 a‘(cid:2)-shaped’secondarystructure(s)duringPCRandwenamed transformantswerescreenedwithaforwardprimer(F1)onthe thisstrategy‘(cid:2)-PCR’basedonthissecondarystructureformed vector and a reverse primer (R1-2) within the target Den inthereaction.The(cid:2)-PCRtechniqueenablespreciselongse- (Fig. 1A), and the rate of positive colonies was about 95% quence modifications including substitution, deletion and in- (Table1;SupplementaryFig.S1A). sertion. In this report, we demonstrate the feasibility and application of (cid:2)-PCR in the generation of constructs for ex- Deletion :-PCR pressingfusionproteins,swappingfluorescenttagsandremov- The principle of deletion (cid:2)-PCR is illustrated in Fig. 2A. The ing subcellular sorting signals. This technology has broad pBI-Den:OsRac3 vector resulting from the substitution (cid:2)-PCR implications for protein engineering, in vitro gene splicing was used as template to test the deletion mode of (cid:2)-PCR. In andgenefunctionanalysis. thiscase,only complementarychimeric primerswereapplied. When the two portions of the chimeric primers annealed to theircomplementarysitesonthetargetconstruct,theOsRac3 Results coding region in the template formed a (cid:2)-shaped structure, The (cid:2)-PCR strategy comprises three modes (insertion/substi- andtheOsRac3-containingloopregionofthe(cid:2)-shapedstruc- tution/deletion);theirprinciplesareillustratedinFigs.1–3.To ture was removed in the de novo PCR product (Fig. 2A, B). demonstratethefeasibilityandrobustnessof(cid:2)-PCR,apBI221- AftertreatmentwithDpnI,thePCRproductwastransformed basedconstruct(pBI-GFP:OsRac3)fortransientexpressionofa into E. coli competent cells. The resultant pBI-Den transfor- fusion of green fluorescent protein (GFP) with OsRac3 mants were screened using a pair of primers, F1/R2, flanking (GFP:OsRac3), a plasma membrane-localized small GTPase the deletion site (Fig. 2A; Supplementary Fig. S1B), and the from rice (Oryza sativa L.) (Chen et al. 2010b), served as the positiveratewasabout100%(Table1). original construct for subcellular localization assays and was Insertion :-PCR modified by substitution, deletion and insertion modes of (cid:2)-PCR,insuccession. The principle of insertion (cid:2)-PCR is illustrated in Fig. 3A. The pBI-Den vector resulting from deletion (cid:2)-PCR was further Substitution :-PCR modified using the insertion mode. Similar to substitution, Theprincipleofsubstitution(cid:2)-PCRisillustratedinFig.1A.We the target Rer1B being inserted was amplified from a Rer1B- containing template with chimeric primers in the first PCR designedapairofchimericprimersforreplacementofGFPwith (Fig. 3B). In the second PCR, the denatured strands of the the gene encoding the photoactivatable fluorescent protein Dendra2 (Den) (Lippincott-Schwartz and Patterson 2009). Rer1B-containingPCRproductsservedasmegaprimersanneal- The 50 sequences oftheforward chimeric primer (Vec-Den-F) ingtotheflankingsequencesoftheinsertionsiteontheplas- andthereversechimericprimer(Vec-Den-R)wereidenticalto mid to form a (cid:2)-shaped structure. Thereby, Rer1B was the flanking sequences of the starting plasmid, while the 30 integrated into the target constructs. After treatment with parts of these primers were identical to the 50 end and 30 DpnI,thePCRproductwastransformedintoE.colicompetent end of the Den coding sequence, respectively (Fig. 1A; cells.ThepBI-Den:Rer1Btransformantswerescreenedbyapair ofprimers,F3/R2(Fig.3A;SupplementaryFig.S1C),andthe Supplementary Table S1). In the first PCR, the target Den positiveratewasabout93%(Table1). fragment was amplified from a Den-containing template with the chimeric primers. Two tails identical to the flanking One-tube :-PCR sequence were integrated in the resultant PCR product (Fig. 1A, B, first lane). In the second PCR, the destination Tosimplifytheproceduresforsubstitutionandinsertionmodes, vector pBI-GFP:OsRac3 served as the template and the dena- we applied exonuclease I to the (cid:2)-PCR, so that these modes tured strands of the Den-containing PCR product served as could be executed using a one-tube method. In the two-step megaprimers annealing to the complementary sequence of protocol,thefirstPCRwasperformedwith28–30cycles,and2– the destination vector through the two flanking tails. The 3ml of the first PCR product was suppliedas megaprimers for (cid:2)-shaped structure in the megaprimer was then extended by thesecondreaction(20ml).Wefoundthattheamountofthe high-fidelityDNApolymerasealongthevectorduringthermo- megaprimers used in the second reaction was equivalent to cycling.Thereby,theGFPfragmentwasreplacedbythetarget that generated by about 12–15 cycles of the first-round PCR Den fragment in the de novo circular plasmid with two stag- (Fig. 4A). Since exonuclease I digests only single-stranded gerednicksattheend(Fig.1A,B,secondlane).Thetemplate DNA without affecting double-stranded DNA, after 12–15 PlantCellPhysiol.54(4):634–642 (2013) doi:10.1093/pcp/pct009 !TheAuthor2013. 635 L.Chenetal. cycles of amplification we added this enzyme to the PCR to remove the remaining chimeric primers, and added 5–10ng oftheplasmidtobemodifiedforfurtheramplification.Inthis way we obtained the expected constructs with substituted or insertedtargetfragmentsinone-tubereactions(Fig.4B). Capacity and efficiency of the :-PCR strategy To test the capacity and efficiency of the (cid:2)-PCR strategy, we used different modes of (cid:2)-PCR for sequence modification of two other gene fragments: OsGEN-L (1.89kb) (Moritoh et al. 2005) and Pi-ta cDNA (2.787kb) (Bryan et al. 2000), and two vectorbackbones:pENTR(2.6kb)(Invitrogen)andpYLTAC747 (15.8kb)(Linetal.2003).Theresultsshowedthat(cid:2)-PCRcan handle large plasmids with sizes ranging from 5.4 to 16.5kb (Fig.5,Table1). Sequence and functional validation of constructs modified by :-PCR strategies We sequenced the junction regions in the three vector con- structsmodifiedinsuccessionbythe(cid:2)-PCRstrategies.Adia- gram and the sequences of the three junction regions in the originalvectorpBI-GFP:OsRac3areshowninFig.6AandB,and these regions were re-sequenced for verification after modifi- cation(Fig.6C–E).Wefoundthatallthreeresultantplasmids had been correctly modified. These plasmids were transiently expressed in rice protoplasts to confirm the subcellular local- izations of the resultant proteins in vivo (Fig. 7). Indeed, Den:OsRac3 (pBI-Den:OsRac3) was localized in the plasma membrane like the original GFP:OsRac3 (pBI-GFP:OsRac3). The Den (pBI-Den) fluorescent protein alone was distributed inthecytoplasmandDen:Rer1B(pBI-Den:Rer1B)waslocalized intheGolgi,asexpected.AlloftheDenproteinsinthedifferent constructs were able to be photoactivated by a 405nm laser andproperlyconvertedfromgreentored(Fig.7). Discussion Although a number of PCR-based cloning and mutagenesis approaches have been reported, most of them are useful for Fig.1Continued 30 junctionofthemodificationsite2(ms2)andthe30 portion(brown) correspondstothe30regionoftheDenfragment.Thedenaturedstrands ofthefirstPCRproductcontainingDen(brown)serveasdenovo‘mega- primers’forsubstitution(cid:2)-PCRinthefollowingreaction.Tosimplifythe Fig.1 Substitution(cid:2)-PCR.(A)Principleofsubstitutionmode(cid:2)-PCR. figure,onlytheforwardmegaprimerandonestrandoftheplasmidtem- Theoriginalconstruct(pBI-GFP:OsRac3)istheplasmidtobemanipu- plate are shown in the preceding steps and figures. When the ‘mega- lated. The GFP fragment (green) is a DNA fragment intended to be primer’annealstothetemplate,thesequencesofGFP(green)andDen replacedbyaphotoactivatablefluorescentproteingeneDen(brown) (brown)formindividual(cid:2)-shapedstructures.(B)TheGFPsequenceinthe ataspecificsiteintheoriginalconstruct.Thechimericforwardprimer pBI-GFP:OsRac3 construct was replaced by Den through substitution (Vec-Den-F)consistsoftwoparts:the50 portion(red)isidenticalto (cid:2)-PCR, resulting in a new construct, pBI-Den:OsRac3. Lane 1, the first thevectorbackbonesequenceatthe50junctionofmodificationsite1 run PCR product Den; lane 2, the second run PCR product (ms1) and the 30 portion (brown) is identical to the 50 region of pBI-Den:OsRac3; lane 3, the starting plasmid pBI-GFP:OsRac3 digested Den; the chimeric reverse primer (Den-Rac3-R) also consists of two with BamHI; * indicates target product bands; Marker, Trans15K DNA parts:the50portion(blue)correspondstotheOsRac3sequenceatthe ladder(TransGenBiotech). 636 PlantCellPhysiol.54(4):634–642 (2013) doi:10.1093/pcp/pct009 !TheAuthor2013. (cid:2)-PCRforprecisesequencemodification onlyonetypeofsequencemodification;thosethatcanbeused for insertion or substitution can handle only very short se- quences. The (cid:2)-PCR methods described herein can be used to manipulate large sequences, and therefore enable all types of sequence modification. The key principle of our (cid:2)-PCR se- quencemodificationstrategiesistheformationofthecharac- teristic secondary structure between the megaprimers (target PCR products) and templates (plasmids) during the PCR. The formation of this secondary structure is attributable to the specificdesignofthechimericprimers.Severaloverlappingex- tensioncloningprotocolswhichallowonlyinsertionsequence modificationhavebeenreported(Chenetal.2000,Bryksinand Matsumura2010,BondandNaus2012),However,our(cid:2)-PCR methods enable all three types of modification in one-tube reactions. Inthetwo-stepreactions,thechimericprimerscarriedinto thesecondPCRarehighly diluted,and thus donotaffect the (cid:2)-PCR. However, for the one-tube (cid:2)-PCR strategy without exonuclease I treatment, the first PCR product was preferen- tially amplified in the second PCR phase, leading to failure of amplificationofthetargetplasmid(datanotshown).However, reducing the concentration of the chimeric primers often re- sultedinlowamplificationefficiency.Oneofthenovelfeatures oftheone-tube(cid:2)-PCRstrategyistheutilizationofexonuclease Itoremovetheextrachimericprimersinthesecondroundof PCRtoavoidtheseproblems.Therefore,nogelpurificationstep ofthetargetfragmentisrequiredineitherofthestrategies.In addition,(cid:2)-PCRsdonotrequirepost-PCRproceduressuchas restrictionenzymedigestionsandligation. The(cid:2)-PCRtechniquecanbeusedtoinsert,swaporremove anyfragmentatanypositionofacircularplasmidforfunctional analysis. The (cid:2)-PCR insertion mode enables subcloning of a target gene directly into a destination plasmid vector by PCR alone. This strategy overcomes the limitation of conventional restriction–ligation-based cloning methods. The substitution mode of (cid:2)-PCR can be used to exchange fluorescent protein genes,fusiontagsorsubcellularsortingsignals,andthedeletion modecanbeusedtogenerateaseriesoftruncatedgenefrag- Fig.2 Deletion(cid:2)-PCR.(A)Principleofdeletionmode(cid:2)-PCR.OsRac3 ments for deletion assays. It is also a very good choice for is a sequence to be deleted from the starting construct in vitro splicing to remove introns from genomic DNA pBI-Den:OsRac3. The chimeric forward primer Den-Vec-F consists of (gDNA) without the need for reverse transcription to clone twoparts:the50 portion(brown)isidenticaltotheDensequenceat full-length cDNA for long genes with few introns. Using dele- the50regionofthemodificationsite2(ms2)andthe30portion(cyan) tion (cid:2)-PCR, we have successfully obtained a full length Pi-ta isidenticaltothevectorsequenceatthe30regionofthemodification cDNA(2,787bp)fromgDNA(4,250bp)(containingone1.5kb site3(ms3).ThechimericreverseprimerDen-Vec-Riscomplemen- intron,datanot shown). Theonly limitation for(cid:2)-PCRis the tarytothechimericforwardprimerDen-Vec-F.Thedeletionmodeis capacity of the DNA polymerase for amplification of longer conductedinonlyonePCR.Whenthechimericprimerannealstothe DNA fragments (target sequences and plasmid vectors). We template,thedeletedfragmentofOsRac3formsa(cid:2)-shapedstructure, highly recommend using a DNA polymerase of high perform- and ms2 and ms3 link together forming ms2/3. (B) The OsRac3 ance and high fidelity, such as PrimeSTAR, Pfx, Pfu, KOD or sequence in pBI-Den:OsRac3 was deleted via deletion mode (cid:2)-PCR, Phusion, to increase the efficiency and fidelity of the (cid:2)-PCR. resulting in the pBI-Den construct. The starting plasmid pBI-Den:OsRac3 was digested by BamHI; * indicates target product UsingthePrimeSTARTaqDNApolymerase,1.89and2.787kb bands;Marker,Trans15KDNAladder(TransGenBiotech). fragmentsweresuccessfullyinsertedorsubstitutedintotarget plasmids by (cid:2)-PCR (Fig. 5, Table 1). The Den fragment was inserted into a transformation-competent artificial chromo- some (TAC) vector pYLTAC747 with a final size of 16.5kb PlantCellPhysiol.54(4):634–642 (2013) doi:10.1093/pcp/pct009 !TheAuthor2013. 637 L.Chenetal. (Fig.5,Table1),demonstratingthatthecapacityof(cid:2)-PCRis sufficient to handle most common binary vectors (9–14kb) for plant transformation. The efficiency of (cid:2)-PCR is around 105–107c.f.u. mg(cid:3)1 and the positive rate of transformants rangesfrom75%to100%(Table1).Thus,the(cid:2)-PCRtechnique isspeedy,effectiveandinexpensive,andwillbenefitmolecular biologistsbyacceleratingtheefficiencyoffunctionalstudiesof targetgenes. Fig. 4 One-tube (cid:2)-PCR strategy. (A) For the first phase of the one-tube(cid:2)-PCRstrategy,wedeterminedthattheamountoftarget sequencein5mlofproductfroma15-cycleamplificationwasapproxi- matelyequaltotheamountin0.5mlofPCRproductproducedby30 cycles of amplification. Therefore, the total amount of the target product in the 20mlreaction from the 15-cycle amplification in the one-tubestrategywasequaltoapproximately2mlofthefullyampli- fiedproduct added to the second PCR in the two-step strategy. (B) Useofone-tube(cid:2)-PCRforinsertionandsubstitutionmodificationsin plasmid construction. Constructs pBI-Den:OsRac3 (left) and pBI-Den:Rer1B (right) were generated by substitution or insertion mode (cid:2)-PCR using the one-tube method; Marker, Trans15K DNA ladder(TransGenBiotech). Fig. 3 Insertion (cid:2)-PCR. (A) Principle of insertion mode (cid:2)-PCR. Coding sequence of a Golgi sorting protein Rer1B is to be inserted into the starting construct pBI-Den. The 50 portion (brown) of the Fig.3Continued forwardchimericprimerRer1B-Fforinsertionmode(cid:2)-PCRisidenti- fragment is PCR-amplified using Rer1B-F and Rer1B-R and serves as de cal to the 50-flanking sequence of the modification site 2/3 (ms2/3) novo‘megaprimers’forinsertioninthefollowing(cid:2)-PCR.(B)Rer1Bwas andthe30portionoftheforwardprimer(purple)isidenticaltothe50 clonedintoavectorcontainingtheDengenethroughinsertion(cid:2)-PCR, end of insert Rer1B. The 50 portion (cyan) of the reverse chimeric resultinginafusedGolgimarkerpBI-Den:Rer1B.Lane1,thePCRproduct primerRer1B-Rcorrespondstothe30-flankingsequenceofthems2/ ofRer1B;lane2,thesecondPCRproductcontainingpBI-Den:Rer1B;lane3, 3 in the vector and the 30 portion (purple) of the reverse primer thestartingplasmidpBI-Den,whichwasdigestedwithBamHI;*indicates corresponds to the 30 end of the insert Rer1B. A Rer1B-containing targetproductbands;Marker,Trans15KDNAladder(TransGenBiotech). 638 PlantCellPhysiol.54(4):634–642 (2013) doi:10.1093/pcp/pct009 !TheAuthor2013. (cid:2)-PCRforprecisesequencemodification Table 1 Capacity and efficiency of the (cid:2)-PCR strategy Mode of :-PCR Final plasmid name Final plasmid Starting plasmid Sub/Del/Ins Positive rate size (kb) size (kb) size (kb) Sub pBI-Den:OsRac3 7.5 7.5 0.717 94.7% Del pBI-Den 6.9 7.5 0.645 100% Ins pBI-Den:Rer1B 7.5 6.9 0.585 93.0% Ins pBI-Den:GEN-L 8.8 6.9 1.89 94.4% Sub pENTR-cPita 5.4 3.3 2.787 93.7% Ins pYLTAC747-Den 16.5 15.8 0.717 78.5% Competentcell efficiency testedby pUC19 was9.3(cid:4)109c.f.u.mg(cid:3)1. Sub,substitution; Del, deletion; Ins,insertion. Positive rate wascalculated basedon thecolony PCRresults of120–150 random clones. modificationsite1(ms1)onthevector,anda21base30portion identical to the 50 end of the insert Den. The 50 portion (20 bases) of reverse primer Den-Rac3-R corresponded to the 50 end of OsRac3, and the 30 portion (21 bases) of Den-Rac3-R correspondedtothe30 endoftheinsertDen(Supplementary TableS1). Primers for deletion of OsRac3. The primers for deletion mode (cid:2)-PCR also consisted of two parts demarcating the ms2 and ms3. The 50 portion of the forward primer Den-Vec-F consisted of 18 bases and was identical to the 50-flanking sequence of the ms2, and the 30 portion (25 bases) of Den-Vec-F was identical to the 30-flanking sequence of the ms3. The reverse primer Den-Vec-R was reversed and complementary to the forward primer Den-Vec-F (SupplementaryTableS1). Primers for insertion of Rer1B. The 50 portion (22 bases) of forwardprimerRer1B-Fforinsertionmode(cid:2)-PCRwasidentical to the 50-flanking sequence of the ms2/3 in the plasmid pBI-Den, and the 30 portion of the forward primer consisted of21basesandwasidenticaltothe50endofinsertRer1B.The50 portionofthereverseprimerRer1B-Rconsistedof21basesand correspondedtothe30-flankingsequenceofthems2/3inthe vector,andthe30portionofthereverseprimerconsistedof21 bases and corresponded to the 30 end of the insert Rer1B (SupplementaryTableS1). Fig.5 Capacityofthe(cid:2)-PCRstrategy.Plasmidsofvarioussizeswere Two-step :-PCR manipulated by (cid:2)-PCR. A nuclear marker GEN-L (1.89kb) was in- serted into pBI-Den plasmid (6.9kb); cPita, a cDNA of resistance For(cid:2)-PCRinsertionandsubstitutionmodes,(cid:2)-PCRcouldbe gene Pi-ta (2.787kb) was substituted with GFP in the pENTR-GFP conductedinatwo-stepreaction.Inthefirstreaction,thePCR plasmid (3.3kb); a photoactivatable fluorescent protein gene Den mixture contained 4.0ml of 5(cid:4) PS buffer, 0.2mM dNTPs, (0.717kb)wasinsertedintothepYLTAC747plasmid(15.8kb).*indi- 0.5mM each of the chimeric primers, and 1–5ng of template catestargetproductbands;Marker,Trans15KDNAladder(TransGen DNA (such as plasmid DNA or gDNA) containing the target Biotech). insertion sequence or substitution fragment, 0.3U of PrimeSTAR Taq DNA polymerase (TAKARA) and deionized water to a final volume of 20ml. A Taq DNA polymerase that Materials and Methods producesblunt-endproductsmustbeusedforthistechnique. ThePCRmixturewassubjectedto28–30cyclesof96(cid:5)Cfor20s, Design of :-PCR primers 60(cid:5)C for 30s, 72(cid:5)C for 1–5min (according to the size of the Primers for substitution of GFP with Den. Forward primer amplified fragment, approximately 45–60s kb–1). In the Vec-Den-F for substitution mode (cid:2)-PCR consisted of a 21 second-step PCRs, 2–3ml of the PCR product resulting from base 50 portion identical to the 50-flanking sequence of the the first reaction served as the megaprimer, and 5–10ng of PlantCellPhysiol.54(4):634–642 (2013) doi:10.1093/pcp/pct009 !TheAuthor2013. 639 L.Chenetal. Fig.6 Validationofthemodifiedconstructsbysequencing.(A)SchematicdiagramoftheoriginalconstructpBI-GFP:OsRac3usedformanipu- lation.Insubstitutionmode,GFPwasreplacedbyDen,whichencodesaphotoconvertiblefluorescentproteinDen,resultinginpBI-Den:OsRac3. OsRac3wasthenremovedfrompBI-Den:OsRac3,resultinginpBI-Den.Rer1BencodingaGolgiproteinwasinsertedintothe30regionofDenusing insertionmode,resultinginpBI-Den:Rer1B.Junctions1–3representtheflankingregionsofthemodificationsites(ms).(B)Originalsequencesof thethreejunctionregionsofpBI-GFP:OsRac3shownin(A).(C–E)Sequenceconfirmationofthejunctionregionsinthemodifiedconstructs producedbydifferent(cid:2)-PCRmodes.PrimersF1–F4indicatedin(A)wereusedforsequencinganalysis.Thearrowheadsindicatethemodifi- cationsites. 640 PlantCellPhysiol.54(4):634–642 (2013) doi:10.1093/pcp/pct009 !TheAuthor2013. (cid:2)-PCRforprecisesequencemodification reactions.PCRmixturecontentsandconditionswereasfollows: 4.0mlof5(cid:4)PSbuffer,0.20mMdNTP,0.5mMofeachchimeric primer,1–5ngoftheplasmidbeingmodifiedastemplate,0.5U of PrimeSTAR Taq DNA polymerase (TAKARA) and deionized watertoafinalvolumeof20ml.Fivecyclesof96(cid:5)Cfor30s,55(cid:5)C for1min,68(cid:5)Cfor5–10min(accordingtothefinalsizeofthe plasmid, approximately 1in kb–1), and 15 cycles of 96(cid:5)C for 1min,68(cid:5)Cfor5–10minwerecarriedout.ThefinalPCRprod- uctsweretreatedat37(cid:5)Cfor30minwith5–10UofDpnI. Sequence validation of modified constructs TheDpnI-treatedfinalPCRproductsweredialyzedagainst0.3(cid:4) TEbufferand1–2mloftheproductsweretransferredintoE.coli (DH5a or DH10B) competent cells by electroporation. Resultant colonies were screened by colony PCR with specific Fig. 7 Subcellular localization offusion proteins expressed fromthe plasmids manipulated by (cid:2)-PCR techniques. GFP:OsRac3 (pBI- primer pairs (Supplementary Table S1). The modified plas- GFP:OsRac3) was localized in the plasma membrane (upper left), as midswereextractedfrompositivecloneswithaplasmidpuri- was Den:OsRac3 (pBI-Den:OsRac3, upper right); Den (pBI-Den) was fication kit (Qiagen) and sequenced with specific primers localized in the cytoplasm (bottom left); Den:Rer1B (pBI-Den:Rer1B) (SupplementaryTableS1). waslocalizedintheGolgi(bottomright).TheDensignalintheovalor circularregionsoftherightpanelswasconvertedfromgreentoredby Transient assays of modified vectors photoactivationwitha405nmlaser.Thesignalswerecollectedsep- Preparationofriceprotoplastcellsandtransfectionwithplas- aratelyandmergedusingtwopseudocolors.Bars=5mm. mids were carried out as described previously (Chen et al. 2010a). Confocal microscopy was performed with an LSM710 the plasmid being modified served as template; PCR buffer, (Carl Zeiss). A 488nm laser was used to excite non- dNTPs and Taq DNA polymerase were present in the same photoconverted Den (detection at 500–550nm, green); Den amounts as in the first reaction. The PCRs were conducted was photoactivated by a 405nm laser and excited by 543nm with five cycles of 96(cid:5)C for 30s, 55(cid:5)C for 1min, 68(cid:5)C for lasers(detectionat550–670nm,red).ThetwosignalsfromDen 5–10min (according to the final size of the plasmid, approxi- werecollectedseparatelyinsequentialmodeandmergedwith mately1minkb–1),followedby10–15cyclesof96(cid:5)Cfor1min, theuseoftwopseudocolors(greenandred). 68(cid:5)Cfor5–10min.TheresultantfinalPCRproductsweretrea- tedat37(cid:5)Cfor30minwith5–10UofDpnItodigestspecifically theoriginaltemplateplasmids. Supplementary data One-tube :-PCR SupplementarydataareavailableatPCPonline. Since current PCR machines possess very powerful program- ming capability, (cid:2)-PCR steps can be performed in the same Funding tubewithoneprogramusingthelinkorinsertionfunctionof PCR machines such as ABI PE9700 or TAKARA TP650. In the This work was supported by the National Natural Science one-tube (cid:2)-PCR, the PCR mixture contained 4.0ml of 5(cid:4) PS Foundation of China [31071646 and 31171350]; the Talent buffer, 0.25mM dNTPs, 0.5mM each of the chimeric primers, Introduction Projects of Guangdong Provincial Colleges and 1–5ng of template DNA containing the target insertion or Universities(2011)[toL.C.]. substitutionfragment,0.5UofPrimeSTARTaqDNApolymer- ase(TAKARA)anddeionizedwatertoafinalvolumeof20ml. Afteramplificationoftheinsertionorsubstitutionfragmentby Acknowledgments 12–16 cycles of96(cid:5)Cfor20s, 60(cid:5)Cfor30s,72(cid:5)Cfor1–5min, We thank Drs. 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