分子生物学DNA测序

DNADNA•••DNA•SangerSangerF,MicklenS,CoulsonAR.1977DNAsequencingandchainterminatinginhibitors.ProcNatlAcadSciUSA,74:5463-5467.•Maxam-GilbertMaxamAMandGilbertW.1977AnewmethodforsequencingDNA.ProcNatlAcadSciUSA,74:560-564.Maxam-GilbertDNAWalterGilbertNobelPrizeChemistry1980•DNADNADNA•—DNA5’—DNAG,A(someG),T(someC),C.—DNA—DNA—•DNADNADNA•SangerNobelPrizeChemistry1980•DNAddNTPddBTP3’dNTP5’-ddNTP3’DNADNA•ddNTPSangerDidesoxynucleotideTriphosphates(ddNTPs)ddATPddGTPddCTPddTTP•—DNA——DNA—dNTPs(dATP,dGTP,dCTP,dTTP)—ddNTPs(ddATP,ddGTP,ddCTP,orddTTP)•—DNAdNTPddNTP—DNA——DNADNADNA•DNA••AGCT+AAGCTAGGTTCATAGGTAAGCTAGGTTCATAGGTAAGCTAGGTTCATAGGTAAGCTAGGTTCATAGGTATCGATCCAAGTCGA+reaction1reaction2reaction3reaction4polymerasepolymerasepolymerasepolymeraseTCGATCCTCGATCCAAATCGATCCAAGTATCGATCCAAGTATCCAAGCT+GTCGA+AGCT+CTCGA+AGCT+TTCGA+TCGATCTCGATCCTCGATCCAAGTATCTCGATCCAAGTATCCTCGATTCGATCCAAGTTCGATCCAAGTATTCGATCCAAGTATCCATAGCT+AAGCTAGGTTCATAGGTATCGA+onereactionpolymeraseTTCGATCCTCGATCCAAATCGATCCAAGTATCGATCCAAGTATCCATCGATCCAAGTCGATCCAAGTATCTCGATCCAAGTATCCTCGATTCGATCCAAGTTCGATCCAAGTATTCGATCCAAGTATCCATTCGATCTCGATCCGC•••••PCR(CycleSequencing)72C•DNAPCR•PCR——DNA—DNA0.51.0mg—3540—50-65C95C50-65C72C95C72C50-65C95CAutomaticSequencerCapillarySequencerDNA••••