Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria 英文参考文献.docVIP

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Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria 英文参考文献.doc

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Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria 英文参考文献

OptimizingTaqPolymeraseConcentrationforImproved Signal-to-NoiseintheBroadRangeDetectionofLow AbundanceBacteria RudolphSpangler1*,NoelL.Goddard2,DavidS.Thaler1,3 1Sackler Laboratory of Molecular Genetics and Informatics, The Rockefeller University, New York, New York, United States of America, 2Department of Physics Astronomy,HunterCollege,CityUniversityofNewYork,NewYork,NewYork,UnitedStatesofAmerica,3HowardHughesMedicalInstitute,AlbertEinsteinCollegeof Medicine,Bronx,NewYork,UnitedStatesofAmerica Abstract Background: PCRinprinciple candetectasingletargetmoleculeinareactionmixture.ContaminatingbacterialDNAin reagentscreatesapracticallimitontheuseofPCRtodetectdilutebacterialDNAinenvironmentalorpublichealthsamples. ThemostpernicioussourceofcontaminationismicrobialDNAinDNApolymerasepreparations.Importantly,allcommercial Taq polymerase preparations inevitably contain contaminating microbial DNA. Removal of DNA from an enzyme preparationisproblematical. Methodology/Principal Findings: This report demonstrates that the background of contaminating DNA detected by quantitativePCRwithbroadhostrangeprimerscanbedecreasedgreaterthan10-foldthroughthesimpleexpedientofTaq enzymedilution,withoutalteringdetectionoftargetmicrobesinsamples.Thegeneralmethodis:Foranythermostable polymeraseusedforhigh-sensitivitydetection,doadilutionseriesofthepolymerasecrossedwithadilutionseriesofDNA orbacteriathatworkwellwiththetestprimers.Forfurtherworkusetheconcentrationofpolymerasethatgavetheleast signal in its negative control (H2O) while also not changing the threshold cycle for dilutions of spiked DNA or bacteria comparedtohigherconcentrationsofTaqpolymerase. Conclusions/Significance:Itisclearfromthestudiesshowninthisreportthatastraightforwardprocedureofoptimizing theTaqpolymeraseconcentrationachieved‘‘treatment-free’’attenuationofinterferencebycontaminatingbacterialDNAin Taqpolymerasepreparations.Thisprocedureshouldfacilitatedetectionandquantificationwithbroadhostrangeprimers ofasmallnumberofb