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1、DNA Sequencing,DNA 序列测定,第一节 Maxam-Gilbert DNA 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
2、C 90C, NaOH (1.2M)， 断裂反应,哌啶（90 C, 1M) 在修饰位点两端使DNA 的糖-磷酸链发生裂解,AC G T+C C,A A T G G C G C C C A C T T C,2. Procedure,Templates,Chemical reaction,a defined DNA restriction fragmentradioactively labeled at one end with 32P,Running PAGE (7M urea) 6 % 20%,Cleavages at specific bases,Autoradiography,AC G T
3、+C C,A A T G G C G C C C A C T T C,4. Notesspecificitylength (200-250bp )it is better to read Maxam and Gilberts article before action,第二节 Sanger dideoxy-mediated chain-termination method,1. Core principle,DNA synthesis will be terminated when a ddNTP is integrated,P,OH,O,H,P,OH,H,P,H,H,3AGCTCGTAGCA
4、TGCATCGATGCATGCTAGCAT 5,dATP, dCTP, dGTP, dTTP,ddATP, ddCTP, ddGTP, ddTTP,Template,dITP,3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5,dATP, dCTP, dGTP, dTTP,ddATP, ddCTP, ddGTP, ddTTP,Template,dITP,3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5,dATP, dCTP, dGTP, dTTP,ddATP, ddCTP, ddGTP, ddTTP,C,C,C,C,C,C,Template,dITP,
5、C,3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5,dATP, dCTP, dGTP, dTTP,ddATP, ddCTP, ddGTP, ddTTP,CA,CA,CA,CA,CA,CA,Template,dITP,CA,3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5,dATP, dCTP, dGTP, dTTP,ddATP, ddCTP, ddGTP, ddTTP,CAT,CAT,CAT,CAT,CAT,CAT,Template,dITP,CAT,3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5,dATP, dCTP, d
6、GTP, dTTP,ddATP, ddCTP, ddGTP, ddTTP,CATC,CATC,CATC,CATC,CATC,CATC,Template,dITP,CATC,3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5,dATP, dCTP, dGTP, dTTP,ddATP, ddCTP, ddGTP, ddTTP,CATCG,CATCG,CATCG,CATCG,CATCG,CATCG,Template,dITP,CATCG,3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5,dATP, dCTP, dGTP, dTTP,ddATP, ddCTP,
7、 ddGTP, ddTTP,CATCGT,CATCGT,CATCGT,CATCGT,CATCGT,CATCGT,Template,dITP,CATCGT,3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5,dATP, dCTP, dGTP, dTTP,ddATP, ddCTP, ddGTP, ddTTP,CATCGTA,CATCGTA,CATCGTA,CATCGTA,CATCGTA,CATCGTA,Template,dITP,CATCGTA,3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5,dATP, dCTP, dGTP, dTTP,ddATP, d
8、dCTP, ddGTP, ddTTP,CATCGTA,CATCGTAC,CATCGTAC,CATCGTAC,CATCGTAC,CATCGTAC,Template,dITP,CATCGTAC,3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5,dATP, dCTP, dGTP, dTTP,ddATP, ddCTP, ddGTP, ddTTP,CATCGTA,CATCGTACG,CATCGTAC,CATCGTACG,CATCGTACG,CATCGTACG,Template,dITP,CATCGTACG,3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5,dA
9、TP, dCTP, dGTP, dTTP,ddATP, ddCTP, ddGTP, ddTTP,CATCGTA,CATCGTACGT,CATCGTAC,CATCGTACG,CATCGTACGT,CATCGTACGT,Template,dITP,CATCGTACGT,3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5,dATP, dCTP, dGTP, dTTP,ddATP, ddCTP, ddGTP, ddTTP,CATCGTA,CATCGTACGTA,CATCGTAC,CATCGTACG,CATCGTACGT,CATCGTACGTA,Template,dITP,CATCG
10、TACGTA,3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5,dATP, dCTP, dGTP, dTTP,ddATP, ddCTP, ddGTP, ddTTP,CATCGTA,CATCGTACGTAG,CATCGTAC,CATCGTACG,CATCGTACGT,CATCGTACGTA,Template,dITP,CATCGTACGTAG,3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5,dATP, dCTP, dGTP, dTTP,ddATP, ddCTP, ddGTP, ddTTP,CATCGTA,CATCGTACGTAG,CATCGTAC,C
11、ATCGTACG,CATCGTACGT,CATCGTACGTA,Template,dITP,CATCGTACGTAGC,A: dNTP + ddATP,G: dNTP + ddGTP,C: dNTP + ddCTP,T: dNTP + ddTTP,ACGT,2. Related strategy,Incorporation,-32P dATP , -33P dATP, -35S dATP,End-labeled primers, -32PrATP,(1) Labeling,(2) Template,M13mp & phagemid,plasmid,ds DNA,ss DNA prepared
12、from,(3) Primer,position,universal primers,Universal(Forward), Reverse, T7, T3, SP6 etc,size, G+C, Tm, degeneration, 2nd structure,E1 S K.S H3,MCS,- 20U,R,-complementation,M13mp18/19, pUC18/19, pGEM series, pBluescript, pTZ series,- 20,MCS,- 20U,R,-complementation,M13mp18/19, pUC18/19, pGEM series,
13、pBluescript, pTZ series,- 40U,R,- 20,- 40,E1 S K.S H3,MCS,- 20U,R,-complementation,M13mp18/19, pUC18/19, pGEM series, pBluescript, pTZ series,- 40U,R,- 20,- 40,- 20U,T3,T7,E1 S K.S H3,Lane A: dNTP + ddATP Lane C: dNTP + ddCTP Lane G: dNTP + ddGTP Lane T: dNTP + ddTTP2 loading,3. Procedure,Full manua
14、l operation,PCR reaction,Automatic sequencing,(1) Full manual operation,United States Biochemical, USB,Sequenase Version 2.0 DNA Sequencing Kit,Brief protocols,Denature templates,65 C to 35 C,Annealing,Labeling,RT 25min,Termination,37C 5min,Running gel,75C 2min, running at 55 C, 2 loading,100C 2min,(ssDNA or ds DNA),stop solution,1530min,
