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1、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
2、(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 CAATGGCGCCCACT
3、TC4.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,d
4、dCTP,ddGTP,ddTTPTemplatedITP3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPTemplatedITP3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCCCCCCTemplatedITPC3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCACACACACA
5、CATemplatedITPCA3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCATCATCATCATCATTemplatedITPCAT3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCCATCCATCCATCCATCCATCTemplatedITPCATC3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddA
6、TP,ddCTP,ddGTP,ddTTPCATCGCATCGCATCGCATCGCATCGCATCGTemplatedITPCATCG3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGTCATCGTCATCGTCATCGTCATCGTCATCGTTemplatedITPCATCGT3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGTACATCGTACATCGTACATC
7、GTACATCGTACATCGTATemplatedITPCATCGTA3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGTACATCGTACCATCGTACCATCGTACCATCGTACCATCGTACTemplatedITPCATCGTAC3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGTACATCGTACGCATCGTACCATCGTACGCATCGTACGC
8、ATCGTACGTemplatedITPCATCGTACG3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGTACATCGTACGTCATCGTACCATCGTACGCATCGTACGTCATCGTACGTTemplatedITPCATCGTACGT3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGTACATCGTACGTACATCGTACCATCGTACGCATCGT
9、ACGTCATCGTACGTATemplatedITPCATCGTACGTA3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGTACATCGTACGTAGCATCGTACCATCGTACGCATCGTACGTCATCGTACGTATemplatedITPCATCGTACGTAG3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP,dCTP,dGTP,dTTPddATP,ddCTP,ddGTP,ddTTPCATCGTACATCGTACGTAGCATCGTAC
10、CATCGTACGCATCGTACGTCATCGTACGTATemplatedITPCATCGTACGTAGCA: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
11、 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-c
12、omplementation 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,US
13、B 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 Se
14、quencing 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 cyclesTTTAAGTGAA
15、AAAACAGCT*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
16、 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 sequ
17、encing 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,pBluesc
18、ript,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 phys
19、ical 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