humana press, methods in molecular medicinemolecular diagnosis of genetic diseases 2nd外语英文电子书

《humana press, methods in molecular medicinemolecular diagnosis of genetic diseases 2nd外语英文电子书》由会员上传分享，免费在线阅读，更多相关内容在教育资源-天天文库。

1、METHODSINMOLECULARMEDICINETMMolecularMolecularDiagnosisofDiagnosisofGeneticDiseasesGeneticDiseasesSecondEditionSecondEditionEditedbyEditedbyRobElles,RobElles,PhDPhDRogerMountford,RogerMountford,BSBSCCCH01,1-8,8pgs8/11/0312:28PMPage11OptimizingPCRforClinicalDiagn

2、osisMichaelP.Bulman1.IntroductionThepolymerasechainreaction(PCR)hasrapidlybecomeanessentialtoolwithinthediagnosticlaboratory.Therefore,itiscrucialwhensettingupanewPCR-basedtesttoensurethatthePCRreactioniscarefullydesignedtobeasrobustandreliableaspossible.Usually

3、,littleoptimizationisrequired.However,therearesomeinstanceswhenaparticularregionofDNAprovesdifficulttoamplifybyPCR.Anumberoffactorsareimportanttoconsiderwhenchoos-ingPCRconditions,andthesearediscussedinthischapter.2.MaterialsAnalytical-gradereagentsshouldbeuseda

4、tallstages,unlessotherwisestated.2.1.PCRReactionBuffersAsanalternativetothebuffersuppliedbythemanufacturer,useofeitherofthefollowingbuffersmaybebeneficialinthePCR.2.1.1.Buffer“A”10X1.670mMTris-HCl(pH8.0at25°C).2.166mMammoniumsulfate.3.37mMmagnesiumchloride.4.67µ

5、Methylenediaminetetraaceticacid(EDTA).5.0.85mg/mLbovineserumalbumin(BSA).Filter-sterilizeandstorein1-mLaliquotsat–20°C.ThisisaparticularlyrobustPCRbuffer,whichprovidesagoodyieldofprod-uct,andisexcellentregardlessofthequalityoftheDNAtemplate.From:MethodsinMolecul

6、arMedicine,vol.92:MolecularDiagnosisofGeneticDiseases,SecondEditionEditedby:R.EllesandR.Mountford©HumanaPressInc.,Totowa,NJ1CH01,1-8,8pgs8/11/0312:28PMPage22Bulman2.1.2.Buffer“R”20X1.1.0MTris-HCl(pH9.0at25°C).2.400mMammoniumsulfate.3.30mMmagnesiumchloride.Filter

7、-sterilizeandstorein1-mLaliquotsat–20°C.Thisbufferisusefulfortemplatesthataredifficulttoamplify,suchasthosewithGC-richtracts,andforlongertemplates(800basepairs[bp]+insize).2.2.DeoxynucleotideTriphosphates(dNTPs)PurchaseindividualdNTPsas100mMstocks(e.g.,RocheDiag

8、nosticsUKLtd,Lewes,UK;dNTPset#1969064).Makea2.5-mMworkingstockbycom-bining2.5µLofeachdNTPandadding90µLofwater.StoredNTPsinsmallaliquotsat–20°Candavoidmultiplefreeze-t