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1、polymerasechainreaction (PCR)DefinitionofPCRThebasicprincipleofPCRPCRFrequentlyAskedQuestionsApplicationofPCRDefinitionofPCRPCR(polymerasechainreaction),isunderthecatalyticofDNApolymerase,tomotherchainDNAasatemplate,withspecificprimersforthethestartingpointofextensio
2、n,throughthedegeneration,annealing,extendingsteps,withtheparentchaintemplateDNAreplicatethecomplementaryDNAchildchaininvitro.ItisoneoftheamplificationtechniqueofDNAsynthesisinvitro,canquicklyspecificamplificateanypurposeDNAinvitro.ThebasicprincipleofPCR1.PCRreactionc
3、omponents(1)templateDNA(2)Primer(3)fourkindsofdeoxyribonucleotides(4)DNApolymerase(5)ReactionBuffer,Mg2+2.PCRreactionsbasicsteps(1)Denaturation,ThehydrogenbondingbetweenthetemplatedoublechainDNArupturedbyheating,anddoublechainapartintoasinglechain.(2)Annealling,Asthe
4、temperaturefalls,theprimerandcomplementaryareaoftemplateDNAcombinedintohybridmolecules.(3)Extension,InthepresenceofDNApolymerase,dNTPs,Mg2+,DNApolymerasecatalyticprimersoutspreadaccordingtothe5'to3'direction,synthesisDNAchainthatcomplementarywithtemplateDNAchain.Thea
5、bovethreestepsforacycle,theproductofeachloopcanbeusedasatemplateforthenextcycle.Afterncycles,thepurposeofDNAplusintheformof2n.GGATCTAGCGTATGCTTGAAACCTAGATCGCATACGAACTTTGAACTTTGGATCTA5’5’3’5’3’3’5’3’primer1primer2TemplateDNAPicture1,PCRprimerscombinedwithtemplatePCRFr
6、equentlyAskedQuestionsA.Noamplificationproducts1.Thecycletemperature:denaturationtemperatureandannealingtemperature2.Theprimerdesign3.TheactivityofDNApolymerase4.Theinhibitoryingredients(proteaseandnuclease)5,DNAsamplesB.Thespecificproductandelectrophoresiscoatingsha
7、ped1.TheconcentrationofMg2+2.Adjustthedosageoftheprimer,templates,andpolymerase3.Decreasethecyclenumber4.Increasingtheannealingtemperature,annealingorextensionoftimeApplicationofPCR1.Thegenediagnosisofgeneticdisease2.Thediagnosisofinfectiousdiseases(hepatitisvirus)3.
8、Thecancergenedetection4.Theapplicationofforensicscience(paternitytest)5.DNAsequencing6.Genecloning7.Introductionofgenemutationpoint
