MolecularBiotechnology

SectionIMolecularHybridizationandBlottingTechniqueAphenomenonortechniqueofformingaheteroduplexfromtwocomplementarypolynucleotidestrandsfromdifferentsources(DNAandDNA;DNAandRNA;RNAandRNA)1.MolecularHybridizationRelatedconceptions/principlesDNAdenaturationDNArenaturation

2.ProbeDNAorRNAfragmentlabeledwithradioisotope,biotinorfluorescentisusedtodetectspecificnucleicacidsequencesbyhybridization.DNAprobeRNAprobe3.BlottingTransfer(blot)biologicmacromoleculesseparatedinthegelandfixthemtonitrocellulose/nylonmembranebydiffusion,electro-transferringorvacuumabsorption,thendetectit.1975,EdwenSouthernBlottingiswidelyusedfordetecting

thepresenceofspecificmacro-molecules(proteins,mRNAsorDNAsequences)inamixture.(1)SouthernblottingGenomicDNA(fromtissuesorcells)arecutbyRE,separatedbygelelectro-phoresisanddenaturedinsolution,thentransferredtoanitrocellulosemembranefordetectingspecificDNAsequencebyhybridizationtoalabeledprobe.UsedtoquantitativelyandqualitativelyanalyzegenomicDNA,recombinantplasmid,screeningDNAlibrary.StepsExtraction:genomicDNAfromtissuesorcellsdigestion:cutwithRESeparation:separateDNAfragmentsinthegelDenaturation:DNAfragmentstreatedtoproducesinglestrandDNATransferring:transferDNAtotheNC.Immobilization:heatingNCin80℃for1～2horuvcrosslink(nylonmembrane)Hybridization:NCisexposedtoprobe(biotin/radioisotope/fluorescence)Detection:developcolors/autoradiography/emitfluorescence.ApplicationQuantitativeandqualitativeanalysisofgenomicDNA,e.g.localizationordetectionofspecificgeneingenome.Analyzetherecombinantplasmidandbacteriophage-screeningDNAlibrary.

(2)Northernblotting

SimilartothesouthernblottingRNAsinsteadofDNAsNoneedofREdigestionApplicationDetectthelevelofspecificmRNAexpressioninsometissuesorcellsComparethelevelofsamegeneexpressionindifferenttissuesorcellsBTBD1andBTBD2Northernblotsofhumantissues.(3)WesthernblottingProteinsamplesareseparatedbyPAGEelectrophoresis,thenelectro-transferredtoNCmembrane.TheproteinsonNCmembranehybridizewithaspecificantibody(1stantibody),thenthetargetproteinbindingwithantibodyisdetectedwithalabeledsecondaryantibody(2nd

antibody).Alsocalledimmunoblotting.ApplicationDetectthespecificprotein;Semi-quantifyspecificprotein;Analyzeproteins’interaction.Protein

HRPDABH2O21Ab2AbHRP:辣根過氧化物酶(horseradishperoxidase)DAB:二氨基聯(lián)苯胺（diaminobenzidine）SectionIIPolymeraseChainReaction(PCR)1.TheInventionofPCRInventedbyKaryMullisin1983.Firstpublishedaccountappearedin1985.AwardedNobelPrizeforChemistryin1993.PCRisatechniqueforamplifyingaspecificDNAsegmentinvitrobymultiplecyclesofDNAsynthesis.ThereactionsystemincludeDNAtemplate,TaqDNA-pol,dNTPs，shortoligonucleotideprimers,buffercontainingMg2+.Theprocessincluding3steps:denaturation,annealing,extension.2.DefinitionTemplateDNA:genomicDNAorplasmidDNAPolymerase:thermostableDNApolymerase,e.g.TaqPrimers:apairofshortoligonucleotides,whichcanhybridizetothetemplateSubstrates:dNTPs(dATP,dCTP,dGTP,dTTP)mustbepresentinequalconcentrations.Reactionbuffer:regulatethePHofthereactionmixtureandsupplyMg2+3.PCRreactionsystemDenaturation:at95℃,doublestrandDNAmeltopentoformsinglestrandDNA.Annealing:55℃±,theprimershybridizewithDNAtemplateExtension:at72℃,Taqpolymerasework,adddNTPstoDNAstrand.4.PCRprocessAbove3stepsareonecycle,repeat25to30cyclesandcanamplifyaspecificDNAsegment.

ThermalCyclersPCRcyclersavailablefrommanysuppliersNotargetproductsaremadeuntilthethirdcycle5.ApplicationofPCRCloningoftargetgeneInvitromutationofgeneMicroanalysisofDNAandRNADNAsequencingGenemutationanalysis6.DerivativePCRRT-PCR(reversetranscriptionPCR)realtimePCR(1)RT-PCRCombinationofRNAreversetranscriptionandPCRreaction.Excellence:quantitativeandqualitativeanalysisofRNA.(2)RealtimePCRQuantitativePCRapairofspecialprimer(labeledfluorescencegroup,RandQ)asprobesInarealtimePCRassay,apositivereactionisdetectedbyaccumulationofafluorescentsignal.TheCt(cyclethreshold)isdefinedasthenumberofcyclesrequiredfor