基因操作原理06

1、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 C 90C, NaOH (1.2M)， 断裂

2、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 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 specificit

3、y 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,

4、 ddTTPTemplatedITP3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP, dCTP, dGTP, dTTPddATP, ddCTP, ddGTP, ddTTPTemplatedITP3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP, dCTP, dGTP, dTTPddATP, ddCTP, ddGTP, ddTTPCCCCCCTemplatedITPC3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP, dCTP, dGTP, dTTPddATP, ddCTP, ddGTP, ddTTPCA

5、CACACACACATemplatedITPCA3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP, dCTP, dGTP, dTTPddATP, ddCTP, ddGTP, ddTTPCATCATCATCATCATCATTemplatedITPCAT3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP, dCTP, dGTP, dTTPddATP, ddCTP, ddGTP, ddTTPCATCCATCCATCCATCCATCCATCTemplatedITPCATC3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dA

6、TP, dCTP, dGTP, dTTPddATP, ddCTP, ddGTP, ddTTPCATCGCATCGCATCGCATCGCATCGCATCGTemplatedITPCATCG3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP, dCTP, dGTP, dTTPddATP, ddCTP, ddGTP, ddTTPCATCGTCATCGTCATCGTCATCGTCATCGTCATCGTTemplatedITPCATCGT3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP, dCTP, dGTP, dTTPddATP, ddCTP,

7、 ddGTP, ddTTPCATCGTACATCGTACATCGTACATCGTACATCGTACATCGTATemplatedITPCATCGTA3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP, dCTP, dGTP, dTTPddATP, ddCTP, ddGTP, ddTTPCATCGTACATCGTACCATCGTACCATCGTACCATCGTACCATCGTACTemplatedITPCATCGTAC3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP, dCTP, dGTP, dTTPddATP, ddCTP, ddGTP

8、, ddTTPCATCGTACATCGTACGCATCGTACCATCGTACGCATCGTACGCATCGTACGTemplatedITPCATCGTACG3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP, dCTP, dGTP, dTTPddATP, ddCTP, ddGTP, ddTTPCATCGTACATCGTACGTCATCGTACCATCGTACGCATCGTACGTCATCGTACGTTemplatedITPCATCGTACGT3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP, dCTP, dGTP, dTTPddATP

9、, ddCTP, ddGTP, ddTTPCATCGTACATCGTACGTACATCGTACCATCGTACGCATCGTACGTCATCGTACGTATemplatedITPCATCGTACGTA3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5dATP, dCTP, dGTP, dTTPddATP, ddCTP, ddGTP, ddTTPCATCGTACATCGTACGTAGCATCGTACCATCGTACGCATCGTACGTCATCGTACGTATemplatedITPCATCGTACGTAG3AGCTCGTAGCATGCATCGATGCATGCTAGCAT 5d

10、ATP, dCTP, dGTP, dTTPddATP, ddCTP, ddGTP, ddTTPCATCGTACATCGTACGTAGCATCGTACCATCGTACGCATCGTACGTCATCGTACGTATemplatedITPCATCGTACGTAGCA: dNTP + ddATPG: dNTP + ddGTPC: dNTP + ddCTPT: dNTP + ddTTPACGT2. Related strategy Incorporation -32P dATP , -33P dATP, -35S dATP End-labeled primers -32PrATP(1) Labeling

11、(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-

12、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: d

13、NTP + 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 2mi

14、n, 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 Terminatio

15、n 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 Sequenc

16、ingReady 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

17、, 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-comple

18、mentation 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:

19、 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*TAAATTCCAAAAGCATTATAG