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Enzymatic Primer-Extension with Glycerol-Nucleoside Triphosphates on DNA Templates 英文参考文献.docVIP

Enzymatic Primer-Extension with Glycerol-Nucleoside Triphosphates on DNA Templates 英文参考文献.doc

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Enzymatic Primer-Extension with Glycerol-Nucleoside Triphosphates on DNA Templates 英文参考文献

EnzymaticPrimer-ExtensionwithGlycerol-Nucleoside TriphosphatesonDNATemplates JesseJ.Chen1.,Ching-HsuanTsai1.¤,XinCai1,AllenT.Horhota2,LarryW.McLaughlin2,JackW. Szostak1* 1HowardHughesMedicalInstitute,andDepartmentofMolecularBiologyandCenterforComputationalandIntegrativeBiology,MassachusettsGeneralHospital,Boston, Massachusetts,UnitedStatesofAmerica,2DepartmentofChemistry,MerkertChemistryCenter,BostonCollege,ChestnutHill,Massachusetts,UnitedStatesofAmerica Abstract Background:Glycerolnucleicacid(GNA)hasanacyclicphosphoglycerolbackbonerepeat-unit,butformsstableduplexes based on Watson-Crick base-pairing. Because of its structural simplicity, GNAis of particular interest with respect to the possibilityofevolvingfunctionalpolymersbyinvitroselection.Template-dependentGNAsynthesisisessentialtoanyGNA- basedselectionsystem. Principal Findings: In this study, we investigated the ability of various DNA polymerases to use glycerol-nucleoside triphosphates (gNTPs) as substrates for GNA synthesis on DNA templates. Therminator DNA polymerase catalyzes quantitativeprimer-extensionbytheincorporationoftwoglyceronucleotides,withmuchlessefficientextensionuptofive glyceronucleotides.Steady-statekineticexperimentssuggestedthatGNAsynthesisbyTherminatorwasaffectedbyboth decreasedcatalyticratesandweakenedsubstratebinding,especiallyforpyrimidines.Inanattempttoimprovepyrimidine incorporation by providing additional stacking interactions, we synthesized two new gNTP analogs with 5-propynyl substitutedpyrimidinenucleobases.ThisledtomoreefficientincorporationofgC,butnotgT. Conclusions:WesuggestthatdirectedevolutionofTherminatormightleadtomutantswithimprovedsubstratebinding andcatalyticefficiency. Citation: Chen JJ, Tsai C-H, Cai X, Horhota AT, McLaughlin LW, et al. (2009) Enzymatic Primer-Extension with Glycerol-Nucleoside Triphosphates on DNA Templates.PLoSONE4(3):e4949.doi:10.1371/journal.pone.0004949 Editor:FloydRomesberg,TheScrippsResearchInstitute,UnitedStatesofAmerica ReceivedFebruary2,

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