OmniChange The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons 英文参考文献.docVIP

OmniChange:TheSequenceIndependentMethodfor SimultaneousSite-SaturationofFiveCodons AlexanderDennig.,AmolV.Shivange.,JanMarienhagen,UlrichSchwaneberg* Lehrstuhlfu¨rBiotechnologie,RWTHAachenUniversity,Aachen,Germany Abstract Focusedmutantlibrarygenerationmethodshavebeendevelopedtoimprovemainly‘‘localizable’’enzymepropertiessuch asactivityandselectivity.Currentmulti-sitesaturationmethodsarerestrictedbythegenesequence,requiresubsequent PCR steps and/or additional enzymatic modifications. Here we report, a multiple site saturation mutagenesis method, OmniChange,whichsimultaneouslyandefficientlysaturatesfiveindependentcodons.Asproofofprinciple,fivechemically cleavedDNAfragments,eachcarryingoneNNK-degeneratedcodon,weregeneratedandassembledtofullgenelengthina one-pot-reaction without additional PCR-amplification or use of restriction enzymes or ligases. Sequencing revealed the presenceofupto27differentcodonsatindividualpositions,correspondingto84.4%ofthetheoreticaldiversityofferedby NNK-degeneration. OmniChange is absolutely sequence independent, does not require a minimal distance between mutatedcodonsandcanbeaccomplishedwithinaday. Citation:DennigA,ShivangeAV,MarienhagenJ,SchwanebergU(2011)OmniChange:TheSequenceIndependentMethodforSimultaneousSite-Saturationof FiveCodons.PLoSONE6(10):e26222.doi:10.1371/journal.pone.0026222 Editor:MartinG.Marinus,UniversityofMassachusettsMedicalSchool,UnitedStatesofAmerica ReceivedJuly21,2011;AcceptedSeptember22,2011;PublishedOctober19,2011 Copyright: ? 2011 Dennig 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:FinancialsupportforthisresearchwasprovidedbyBASFSocietasEuropaea(BASFSE)(/group/corporate/en/index).Thefunders hadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript. CompetingInterests:Theauthorshaveread