A One Pot, One Step, Precision Cloning Method with High Throughput Capability 英文参考文献.docVIP

AOnePot,OneStep,PrecisionCloningMethodwith HighThroughputCapability CarolaEngler,RomyKandzia,SylvestreMarillonnet* IconGeneticsGmbH,BiozentrumHalle,Halle,Germany Abstract Currentcloningtechnologiesbasedonsite-specificrecombinationareefficient,simpletouse,andflexible,buthavethe drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressedprotein.WehavedevisedasimpleandrapidsubcloningstrategytotransferanyDNAfragmentofinterestfrom anentrycloneintoanexpressionvector,withoutthisshortcoming.ThestrategyisbasedontheuseoftypeIIsrestriction enzymes,whichcutoutsideoftheirrecognitionsequence.Withproperdesignofthecleavagesites,twofragmentscutby type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called ‘Golden Gate’ cloning was devised that allows to obtain in one tube and one step close to one hundredpercentcorrectrecombinantplasmidsafterjusta5minuterestriction-ligation.Thismethodisthereforeasefficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwantedsequencesinthefinalconstruct,thusprovidingprecisionforthisfundamentalprocessofgeneticmanipulation. Citation:EnglerC,KandziaR,MarillonnetS(2008)AOnePot,OneStep,PrecisionCloningMethodwithHighThroughputCapability.PLoSONE3(11):e3647. doi:10.1371/journal.pone.0003647 Editor:HanyA.El-Shemy,CairoUniversity,Egypt ReceivedAugust23,2008;AcceptedOctober20,2008;PublishedNovember5,2008 Copyright: ? 2008 Engler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding:WorkfundedbyIconGeneticsGmbH. CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:marillonnet@icongenetics.de Introduction DNAoverhangs(dependingontheenzyme)