基因操作原理06.ppt
DNA Sequencing DNA 序列测定序列测定第一节第一节 Maxam-Gilbert DNA化学降解法化学降解法一、基本原理直接或间接特异性识别4 种碱基特定化学试剂可对碱基进行特异性修饰在修饰碱基处(5或3)打断磷酸二酯键Reagents for MG DNA sequencing Base specificity Base reaction C hydrazine+NaCl(1.5M)G dimethyl sulphate(硫酸二甲酯),methylation A+G acid(哌啶甲酸),pH2.0，脱嘌呤 C+T hydrazine(肼)，打开嘧啶环 A C 90C,NaOH(1.2M)，断裂反应哌啶（哌啶（90 C,1M)在修饰位点两端使在修饰位点两端使DNA 的糖的糖-磷酸链发生裂解磷酸链发生裂解 AC G T+C CAATGGCGCCCACTTC2.Procedure Templates Chemical reaction a defined DNA restriction fragment radioactively labeled at one end with 32P Running PAGE(7M urea)6%20%Cleavages at specific bases Autoradiography AC G T+C CAATGGCGCCCACTTC4.Notes specificity length(200-250bp)it is better to read Maxam and Gilberts article before action第二节 Sanger dideoxy-mediated chain-termination method1.Core principleDNA synthesis will be terminated when a ddNTP is integratedPOHOHPOHHPHH3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPTemplatedITP3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPTemplatedITP3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCCCCCCTemplatedITPC3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCACACACACACATemplatedITPCA3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCATCATCATCATCATTemplatedITPCAT3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCCATCCATCCATCCATCCATCTemplatedITPCATC3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGCATCGCATCGCATCGCATCGCATCGTemplatedITPCATCG3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGTCATCGTCATCGTCATCGTCATCGTCATCGTTemplatedITPCATCGT3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGTACATCGTACATCGTACATCGTACATCGTACATCGTATemplatedITPCATCGTA3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGTACATCGTACCATCGTACCATCGTACCATCGTACCATCGTACTemplatedITPCATCGTAC3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGTACATCGTACGCATCGTACCATCGTACGCATCGTACGCATCGTACGTemplatedITPCATCGTACG3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGTACATCGTACGTCATCGTACCATCGTACGCATCGTACGTCATCGTACGTTemplatedITPCATCGTACGT3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGTACATCGTACGTACATCGTACCATCGTACGCATCGTACGTCATCGTACGTATemplatedITPCATCGTACGTA3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGTACATCGTACGTAGCATCGTACCATCGTACGCATCGTACGTCATCGTACGTATemplatedITPCATCGTACGTAG3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGTACATCGTACGTAGCATCGTACCATCGTACGCATCGTACGTCATCGTACGTATemplatedITPCATCGTACGTAGCA:dNTP+ddATPG:dNTP+ddGTPC:dNTP+ddCTPT:dNTP+ddTTPA C G T2.Related strategy Incorporation -32P dATP,-33P dATP,-35S dATP End-labeled primers -32PrATP(1)Labeling(2)Template M13mp&phagemidplasmid ds DNA ss DNAprepared from(3)Primer position universal primersUniversal(Forward),Reverse,T7,T3,SP6 etc size,G+C,Tm,degeneration,2nd structure E1 S K.S H3 MCS-20 UR-complementation M13mp18/19,pUC18/19,pGEM series,pBluescript,pTZ series-20 MCS-20 UR-complementation M13mp18/19,pUC18/19,pGEM series,pBluescript,pTZ series -40 UR-20-40E1 S K.S H3 MCS-20 UR-complementation M13mp18/19,pUC18/19,pGEM series,pBluescript,pTZ series -40 UR-20-40-20 UT3T7E1 S K.S H3 Lane A:dNTP+ddATPLane C:dNTP+ddCTPLane G:dNTP+ddGTPLane T:dNTP+ddTTP2 loading3.Procedure Full manual operation PCR reaction Automatic sequencing (1)Full manual operationUnited States Biochemical,USB Sequenase Version 2.0DNA Sequencing Kit Brief protocols Denature templates65 C to 35 C Annealing Labeling RT 25min Termination 37C 5min Running gel 75C 2min,running at 55 C,2 loading100C 2min(ssDNA or ds DNA)stop solution 1530min(2)PCR reaction(2)PCR reactionCircumVent Thermal Cycle Dideoxy DNA Sequencing Kitwith Vent R (exo-)DNA Polymerase Brief protocols Denature templates(ssDNA or ds DNA)65 C to 1530min Annealing Labeling RT 25min Termination 37C 5min,stop solution Running gel 75C 2min,running at 55 C,2 loading100C 2minP C RPCR procedure95 C 20 sec55 C 20 sec72 C 20 sec20 cyclesTTTAAGTGAAAAAACAGCT*TAAATTCCAAAAGCATTATAGAAGT*ATCAATTT T G C A Primer extension for CTC gene(3)Automatic sequencingABI PRISMdRhodamineTerminatorCycle SequencingReady Reaction KitWith AmpliTaq DNA Polymerase,FSPE Applied Biosystemsa division of Perkin-ElmerCore principleKey technique Labeling ddNTP with 4 kind of dRhodamine Each dRhodamine give different colorA greenC yellowG blueT redPCR procedureReagent Terminator Ready Reaction Mix(Taq,buffer,dNTP,labeled ddNTP)Template primer H2OPCR reaction 96 C 10 sec50 C 5 sec60 C 4min25 cyclesPurification and running gel Ethanol/NaAcPAGEAutomatic analysisfor sequencing a DNA fragment Strategy primer template1.Primer walkingSynthesizing oligo-nucleotides2.Random clones EI S K.S H3 MCS-20 UR-complementation M13mp18/19,pUC18/19,pGEM series,pBluescript,pTZ series -40 UR-20-40-20 UT3T72.Random clones Inserting into pUC18/19M13mp18/19,pUC18/19,pGEM series,pBluescript,pTZ series Sn=1-(1-/L)n-Sn:ratio of covering L:the length of the target DNA:the mean length read at each testing n:the number of needed clonesifthen(Sn=50%,n10)L=8000,=400,Sn=98%n=76ifthen n=101309 L=5,500,000,=500,Sn=99.99%N=log(1-Sn)/log(1-/L)coverage 9.21 times3.Fixed clonedConstructing physical map 4.Deletion clonesErase-a-Base SystemPromegaExonuclease III5353533AGCUCGUAGCAUGCAU5 CATCGTA CATCGTACGTAG CATCGTAC CATCGTACG CATCGTACGT CATCGTACGTA3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5DNARNA CATCGTACGTAPrimer extension TTTAAGTGAAAAAACAGCT*TAAATTCCAAAAGCATTATAGAAGT*ATCAATTT T G C A Primer extension for CTC gene七、RNA端点分析1.S1酶作图标记寡核苷酸,PAGE电泳检测MW53ATGUAGS1酶S1酶还可检测前体RNA-mRNA的剪接点
