Robust one-Tube ?-PCR Strategy Accelerates Precise Sequence Modification of Plasmids for Functional Genomics.

1.2 MB·English

by  Chen, Letian

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,[phone].
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.
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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
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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
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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
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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. Sylvie Maisonneuve and Hann Ling Wong for
5–10UofexonucleaseI(NEB)wasappliedtodigesttheremain-
ingchimericprimersat37(cid:5)Cforabout10min.Then5–10ngof helpfuldiscussions,andXuegangSunfortechnicalassistancein
imaging.
theplasmidtobemodifiedwasaddedasthesecondtemplate,
followedbyfivecyclesof96(cid:5)Cfor30s,55(cid:5)Cfor1min,68(cid:5)Cfor
8min,and15cyclesof96(cid:5)Cfor1min,68(cid:5)Cfor8min.Thefinal
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