Golden Gate Shuffling A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes.doc


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GoldenGateShuffling:AOne-PotDNAShufflingMethod
BasedonTypeIIsRestrictionEnzymes
CarolaEngler.,RamonaGruetzner.,RomyKandzia,*
icsGmbH,BiozentrumHalle,Halle,Germany
Abstract
ubeatleastnineseparateDNAfragmentstogetherintoan
acceptorvector,with90%
oftype IIsrestriction enzymes andis performedbysimplysubjectinga mix of10undigested inputplasmids (nineinsert
eptorvector)toarestriction-
bininginonereactionseveralfragmentsetsprepared
from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three
parentaltemplates(bovinecationictrypsinogen,bovineanionictrypsinogenandhumancationictrypsinogen)eachdividedin

19,binationsinonesinglerestriction-ligationandthatexpressionscreeningofasubsetofthelibrary
,thatwecall‘Golden
Gateshuffling’,isrobust,simpleandefficient,canbeperformedwithtemplatesthathavenohomology,bined
withothershufflingprotocolsinordertointroduceanyvariationinanypartofagivengene.
Citation: Engler C, Gruetzner R, Kandzia R, S (2009) Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction
Enz

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  • 时间2018-08-29