博士学位论文答辩范文

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1、化学与分子工程学院答辩人答辩人:魏魏 芳芳指导老师指导老师:赵新生赵新生2003年年6月月6日日l课题背景l检测设计思想l交流阻抗检测表面杂交l镶嵌型染料检测表面杂交l致谢l碱基互补配对杂交原理Affymetrix p53 chipl DNA序列的突变PMperfect match完全配对序列完全配对序列SMsingle-base mismatch单点错配序列单点错配序列DMdouble-base mismatch两点错配序两点错配序列列l目前基因芯片的检测方法目前基因芯片的检测方法有标记有标记:荧光、同位素标记、酶标法荧光、同位素标记、酶标法无标记无标记:交流阻抗、石英晶体微天平、交流阻抗、

2、石英晶体微天平、表面等离子体共振、质谱法表面等离子体共振、质谱法l目前基因芯片的不足目前基因芯片的不足成本、特异性、全局优化成本、特异性、全局优化 单个碱基不同的寡聚核酸序列的识别单个碱基不同的寡聚核酸序列的识别(SNP)Hairpin design+Potential controlCOOHNHS/EDCMMcomplementarysingle-base mismatchSiOHOSiOHOSiOHOSiTyagi,S.,et al Nature Biotechnology 1996,14,303l改变溶液的浓度、pH、离子强度进行控制l利用温度变化进行控制l利用外加电场外加电场进行控制T

3、omoharu kajiyama,Patent No:US2002164778 Hitachi High-Technologies CorplConcentration Change-accumulationlConformation Change-orientation,stretchlDuplex Change-annealing,denaturation SiMartin Stelzle,et al,Fresenius J Anal Chem(2001)371:112119lConcentration Change-accumulationlConformation Change-ori

4、entation,stretchlDuplex Change-annealing,denaturation SiShana O.Kelley,et al,Langmuir,Vol.14,No.24,1998 6783lConcentration Change-accumulationlConformational Change-orientation,stretchlDuplex Change-annealing or denaturation SiComplementary targetSiHeaton et al.PNAS,2001 vol.98 no.7 3703Si(111)H-ter

5、minatedAlkyl modifiedetchRR RRRHHH HHF/NH4FUV lightAtomic flat monolayer covalently bonded to the Si(111)surface硅表面为-COOH将-COOH表面活化 DNA fragments NHS/EDC complementary DNA sequence NOCOOOCOOHCsingle strand DNAOCOdouble strand DNA活化后的表面与单链DNA反应接有单链DNA的表面与配对链杂交l 交流阻抗识别PM/SMl 镶嵌型染料识别PM/SM01002003004005

6、00600non-complementary strandnon-complementary strandcomplementary strandcomplementary strandcomplementary strandnon-complementary strand delta(C)%Time(second)good discrimination and reusability of hairpin probesRound 1Round 2Round 3探针分子5-GCGAGC CCT CCG GTT CAT GCC GCTCGC-3目标分子：完全匹配(WT)5-GGC ATG AAC

7、 GG AGG-3点错配(MT)5-GGC ATG AAC TGG AGG-3Codon 248 TGAAC C GGAGG 位于第7个外显子上：Exon 7发生点突变的模式发生点突变的模式CGG TGG，编码的氨基酸为 Arg Trp 发卡的环发卡的环from segment of p53网上共享计算软件：MfoldTMZuker,et al.http:/www.bioinfo.rpi.edu/applications/Probe15-GCGAGC CCT CCG GTT CAT GCC GCTCGC-3Probe25-CCT CCG GTT CAT GCC-3Probe35-GCGAGC

8、GTT CAT GCC GCC CAT GCTCGC-3Probe45-CGAGC GTT CAT GCC GCC CAT GCTCG-36565 Tm(WT)(C)Tm(Pc)(C)Tm(MT)(C)Probe 1 68 66 60Probe 2 68 27 60Probe 3 68 68 54Probe 4 68 59 54MTWTDesired:Tm(WT)Tm(Pc)Tm(MT)4 Probes are designed to test our idea on the optimal performancePc-1.0-0.8-0.6-0.4-0.20.001x10132x10133x

9、10134x1013PcPc+MTPc+WT 1/(Cs)2(cm2/F2)Potential(vs SCE)(V)Sample WECE(Pt)RE(SCE)WE0200400600800 10000.940.960.981.000.940.960.981.000.940.960.981.00 -0.3V -0.5VNormalized CapacitanceTime(s)-0.7V21Real time monitor of hybridization1:addition of MT2:addition of WTDiscrimination between WT and MT depen

10、ds on the electric potential applied-1.0-0.8-0.6-0.4-0.20.0-20-10010203040Probe2Probe3Probe4Probe1 D(vs SCE)(V)Best design(Probes1 and 4)gives the highestdiscrimination between WT and MTl本实验中成功的将DNA分子接在了硅电极表面，并可以进行杂交反应和解链反应。l实验所采用的利用阻抗对DNA修饰表面的杂交过程检测及实时检测的灵敏度较高，并且时间响应很快。l合理的探针分子设计与电势控制结合，可以实现对单个碱基错配

11、序列的特异性识别。Intercalator Dye to duplex of DNALerman,L.S.J.Mol.Biol.,1961,3,18-30.Kinetics of Intercalate Initial fast concentration-dependant reactionhydrophobic and electrostatic assist Slow insert process to the intercalate sites of duplex Dissociation is slow and complicatedPicoGreen-Intercalator Dy

12、e to duplex of DNAunsymmetrical cyanine dyeEx 480nm,Em 520nmStable to photobleachingFluorescence Enhancement50055060065070002004006008001000 Fluorescencewavelength(nm)1-Background2-Probe1 only3-Hybridized with SM4-Hybridized with PM1234MMComplementary targetSingle base mismatched targetSiSiHybridiza

13、tion SiPicoGreenDarkPicoGreen探针分子探针分子-LPL(Lipoprotein Lipase sequence containing 447X SNP）发生突变模式发生突变模式:Wild type:TCA 447 SNP:TGA种类DNA序列Probe15-CCCCC GTT TCA GCC TGA CTT CTT ATT GGGGG-3Probe25-GTT TCA GCC TGA CTT CTT ATT-3PM5-AAT AAG AAG TCA GGC TGA AAC-3SM5-AAT AAG AAG TGA GGC TGA AAC-3DM5-AAT AAG T

14、AG TCA GGG TGA AAC-3CElaser 514 nmWECEIn situ fluorescence detection cell发卡型探针分子可以清楚地分辨发卡型探针分子可以清楚地分辨PM，DM和和SM-1.6-1.4-1.2-1.0-0.8-0.6-0.4-0.2 0.00.20.40.6-0.20.00.20.40.60.81.01.2 IntensityPotential(V)HPM HSM HDM-1.0-0.8-0.6-0.4-0.20.00.20.40.6-0.20.00.20.40.60.81.0 LPM LSM LDM IntensityPotential(V

15、)线型探针分子无法区分线型探针分子无法区分PM，SM和和DM解链半高电位解链半高电位 E1/2-1.6-1.4-1.2-1.0-0.8-0.6-0.4-0.20.00.00.20.40.60.81.01.2 IntensityPotential(V)E1/2-2.0-1.5-1.0-0.50.0-0.20.00.20.40.60.81.01.2Data:pt50mVmin_DModel:sigmoidal Equation:y=P1*exp(x-P2)/P3)/(1+exp(x-P2)/P3)Weighting:yNo weighting Chi2/DoF=0.00116R2=0.99402

16、P10.998?.01135P2-0.48801?.00531P30.05399?.00462 10mV/min 20mV/min 50mV/min IntensityPotential(V)PM解链电势扫描曲线SM解链电势扫描曲线-2.0-1.6-1.2-0.8-0.40.00.4-0.20.00.20.40.60.81.01.2Data:mt20mVmin2_DModel:sigmoidal Equation:y=P1*exp(x-P2)/P3)/(1+exp(x-P2)/P3)Weighting:yNo weighting Chi2/DoF=0.00114R2=0.99492 P11.0

17、0064?.02494P2-0.0642?.01064P30.09438?.00877 10mV/min 20mV/min 50mV/min IntensityPotential(V)PM和和SM的解链电位曲线与扫描速度相关性不同的解链电位曲线与扫描速度相关性不同解链电位曲线随扫描速度减慢而负移解链电位曲线随扫描速度减慢而负移Scan rate(mV/min)E1/2-PM(V)E1/2-SM(V)E1/2-DM(V)|E1/2(PM)-E1/2(SM)|(V)10-0.99-0.16-0.090.8320-0.64-0.06-0.5850-0.490.04-0.53l双链解链反应机理l利用解

18、链电位曲线计算现场杂交时的碱基能量差值l如何更好的控制双链的解链-1.6-1.4-1.2-1.0-0.8-0.6-0.4-0.2 0.00.20.40.6-0.20.00.20.40.60.81.01.2 IntensityPotential(V)HPM HSM HDMl采用无标记方法检测，利用PicoGreen与双链结合产生的荧光对DNA片断修饰电极的杂交过程进行监控，降低成本，操作简便。l第一次获得了DNA修饰电极的双链解链电位曲线。此曲线可以观察到“完整”双链的解链过程，从而避免了文献中在静态电位下测量的偶然实验结果。同时可以提供详细的动力学信息。l定义了双链解链电位曲线的“半高电位”作

19、为特异性识别的判据。这一判据的引入可以避免实验误差所带来的假阳性和假阴性结果，使判断更加可靠、重复。l电位扫描法的引入，可以不必探索在芯片点阵上每点的最佳测量条件，直接进行高度平行化的成像技术检测，满足基因芯片高通量检测的需要。lFang Wei,Xin Sheng Zhao THIN SOLID FILMS 408(1-2):286-290 APR 3 2002 lFang Wei,Bin Sun,Yuan Guo,Xin Sheng Zhao BIOSENSORS&BIOELECTRONICS 18(9):1157-1163,2003lFang Wei,Bin Sun,Wei Liao,J

20、ianhua Ouyang,Xin Sheng ZhaoBIOSENSORS&BIOELECTRONICS 18(9):1149-1155,2003l赵新生，魏芳魏芳，孙豳发卡形探针基因芯片及其制备方法和检测方法发卡形探针基因芯片及其制备方法和检测方法 国家发明专利号国家发明专利号 CN1373228lConghua Lu,Nianzu Wu,Fang Wei,et al.ADVANCED FUNCTIONAL MATERIALS,In press,2003lCao TB,Fang Wei,Yang YL,et al.LANGMUIR 18(13):5186-5189 JUN 25 2002

21、lCao TB,Fang Wei,Huang L,et al.NEW J CHEM 26(3):269-271 MAR 2002 lHuang L,Hou XR,He YK,Zheng XL,Fang Wei,Zhao XS,Cao WXCHINESE J POLYM SCI 20(3):197-203 MAY 2002 lHuang L,Fan S,Fang Wei,Xin-sheng Zhao,Jin-xin Xiao and Bu-yao ZhuCHINESE J POLYM SCI 20(5):397-400 MAY 2002lHuang L,Huang F,He YK,Fang Wei,Zhao XS,Cao WXACTA POLYM SIN(2):192-197 APR 2002 l张黎征,魏芳魏芳,赵新生 ACTA PHYS-CHIM SIN 16(4):370-373 APR 2000l指导教师指导教师：赵新生 教授杨华铨 教授曹维孝 教授郭 源 教授l本组成员本组成员：欧阳贱华，廖玮，关英华，翟林，陈旭东，陈春来，徐佳于安池，罗国斌，柳汀汀，吴瑞阁，应丽贞，孙豳，洪莲，张黎征，李密，郭锐，王胜龙，屈军艳，李巍鲍海凌，赵临，林颖，武巍，高婷娟l合作成员：合作成员：曹廷炳，鲁丛华