英文課件 核酸

BIOCHEMISTRYNEUCLEICACIDSBIOCHEMISTRYNEUCLEICACIDS1CONTENTS4.1TwoNucleicAcids:DNAandRNA4.2Propertieofnucleotides核苷酸4.3PrimaryStructureofNucleicAcids核酸的一級(jí)結(jié)構(gòu)4.4SecondaryStructureandtertiaryStructureofNucleicAcids核酸的二級(jí)結(jié)構(gòu)和三級(jí)結(jié)構(gòu)4.5StabilityofSecondaryandTertiaryStructure核酸的二級(jí)結(jié)構(gòu)和三級(jí)結(jié)構(gòu)的穩(wěn)定性CONTENTS4.1TwoNucleicAcids:2英文課件核酸34.2NucleotidesNucleotides=Base+Sugar+Phosphate

Nucleoside=Sugar+Base(nophosphate)PRdRPbasebaseRiboseordeoxyribose(核糖或脫氧核糖）4.2NucleotidesNucleotides=4adenine（腺嘌呤）urecil（尿嘧啶）thymine（胸腺嘧啶）cytosine（胞嘧啶）guanine（鳥嘌呤）PurinebasesPyrimidineBasesadenine（腺嘌呤）urecil（尿嘧啶）thymine5StandardNucleotides：5’-adenosinemonophosphate5’-AMP（5’-腺苷酸）5’-guanosinemonophosphate5’-GMP（5’-鳥苷酸）5’-cytidinemonophosphate5’-CMP（5’-胞苷酸）5’-uridinemonophosphate5’-UMP（5’-脲苷酸）5’-deoxyadenosinemonophosphate5’-dAMP（5’-脫氧腺苷酸）5’-deoxyguanosinemonophosphate5’-dGMP（5’-脫氧鳥苷酸）5’-deoxycytidinemonophosphate5’-dCMP（5’-脫氧胞苷酸）5’-deoxythymidinemonophosphate）5’-dTMP（5’-脫氧胸腺苷酸StandardNucleotides：5’-adeno6英文課件核酸7MinorBasesorModifiedbases

(稀有堿基或修飾堿基)MinorBasesorModifiedbases

8Nucleotidepolyphosphate多磷酸核苷8NDPs（5’-nucleotidediphosphate5’-核苷二磷酸）8NTPs（5’-nucleotidetriphosphate5’-核苷三磷酸）ATPNucleotidepolyphosphate多磷酸核苷89CyclicNucleotidesCyclicNucleotides10CoA、NAD+（P）andFADCo-enzymeA（CoA）Nicotinamideadeninedinucleotide（NAD+）煙酰胺腺嘌呤二核苷酸、FlavinAdenineDinucleotide

（FAD）黃素腺嘌呤二核苷酸CoA、NAD+（P）andFADCo-enzyme11NADNAD12NADPNADP13FADFAD14Nucleotidepropertics1.DissociationinwaterThephosphategroupofanucleotideactsasastrongacid(pKa1),Theaminegroupsofthepurineandpyrimidinebasescanbeprotonated.Nucleotidepropertics1.Dissoc152.TautomerizationofthebasesThebasescantautomerize(互變異構(gòu)）;thatis,thebasescanredistributepositionsofhydrogensanddoublebonds2.Tautomerizationofthebases16Unusualbasepair見于酵母苯丙氨酰tRNAUnusualbasepair見于酵母苯丙氨酰tRNA171-methyladenine=T1-methyladenine=T18Nucleotidepropertics3.SpectrumNucleotidesabsorblightstronglyinthenear-ultravioletregionofthespectrum.Nucleotidepropertics3.Spectr19260（X104）AMP1.54GMP1.17CMP0.75dTMP0.92UMP0.99.260（X104）20Biologicalfunctionsofnucleotides1、Themonomericunitsofnucleicacids2、Energycarrier：ATP、GTP、CTP、UTP3、signals：cAMP、cGMP4、coenzymes：FAD、NAD、CoABiologicalfunctionsofnucleo214.3PrimaryStructureofNucleicAcidsTheprimarystructureisthesequenceofnucleosidemonophosphatesDirectionality：5’3’IndividualitypGGTA——,pGpGpTpA——,GGTA——pGGTA（OH）4.3PrimaryStructureofNucle22SignificanceofPrimaryStructure

GeneticinformationstoredinnucleotidesequenceofDNA

GeneisaparticularDNAsequenceSignificanceofPrimaryStruct23

DNAisgeneticsubstance.Avery,MacLeod,McCartyDNAisgeneticsubstance.Aver24HersheyandChase-BacteriophageT2transfersDNAtobacteriaHersheyandChase-Bacterioph254.4SecondarystructureofDNAThesecondarystructurereferstotheshapeanucleicacidassumesasaresultoftheprimarystructure.B-DNA:predominantformintheaqueousenvironmentofthecellA-DNAZ-DNA:purine/pyrimidinetracts4.4SecondarystructureofDN26SecondarystructureofDNA11/0.23nm10/0.34nm12/0.38nmMajorgrooveMinorgrooveSecondarystructureofDNA11/27

28StructureofB-DNA—Thetwochainsinthedoublehelixareantiparallel—Phosphategroupslinktogetherthesugarbackboneviaphosphodiesterbonds.—Thebasesonthetwochainspairinacomplementaryfashion.A=T，G=C—Hydrogenbondsbetweenbasesholdthedoublehelixtogether.(A=T，GC)—B-DNA:d=2nm，pitch=3.4nm,10bp/turnofthehelixStructureofB-DNA—Thetwo29DNAbasepairDNAbasepair30DNAbasepairDNAbasepair31DNAbasepairPrictice:Writethesequence.DNAbasepairPrictice:32ThediscoveryofDNAdoublehelixChargaff'sRule(A=T,G=CinDNA)Franklin,Wilkins:X-rayDiffraction

RefinedStructureThediscoveryofDNAdoublehe33Watson,CrickWatson,Crick34SignificanceofDNAdoublehelixsemi-conservativemodelofDNAreplicationSignificanceofDNAdoublehel35MeselsonandStahlexperiment1958MeselsonandStahlexperiment136ApplicationofComplementarityDNA半保留復(fù)制核酸分子雜交實(shí)驗(yàn)MoleculehybridizationApplicationofComplementarity37UnusualSecondaryStructuresofDNAPalindromes(回文結(jié)構(gòu)）Cruciformformationinpalindromesequences

GGCGCGCC

CCGCGCGGUnusualSecondaryStructureso38H-DNApurines/pyrimidinesononestrand

H-DNApurines/pyrimidinesonon39英文課件核酸404.5TertiarystructureofDNATertiarystructurereferstolarge-scalefoldinginalinearpolymerthatisatahigherorderthansecondarystructure.Thetertiarystructureisthespecificthree-dimensionalshapeintowhichanentirechainisfolded.4.5TertiarystructureofDNAT41TertiarystructureofDNATertiarystructureofDNA42CircularDNAandSupercoiling

CircularDNAsSupercoiling:Positive(+)----additionaltwistsaddedbeyondthenormalamountforlinearDNAnegative(-)----reducednumbersoftwistscomparedtolinearDNA.CircularDNAandSupercoiling43SupercoilingTwists(T,纏繞數(shù)):Thetotalnumberoftimesthetwostrandsofthehelixcrossovereachother,excludingwrithing.Twistisameasureofhowtightlythehelixiswound.Writhes(W,扭曲數(shù)):Thenumberoftimesthehelixasawholecrossesoveritself-thatis,thenumberofsuperhelicalturnsthatarepresentLinkingnumber(L,連接數(shù)):ThetotalnumberoftimestwostrandsofaDNAhelixcrosseachotherbymeansofeithertwistorwritheL=T+WThesuperhelicaldensity:L/L0,whereL0isthelinkingnumberoftheDNAinitsunstrained(relaxedstate).SupercoilingTwists(T,纏繞數(shù)):44英文課件核酸45Besideswrithing,unwindingDNA,cruciformformation(viapalindromes),triplehelixformation(H-DNA),andZ-DNAformation,canallreducesuperhelicaltension,too.Besideswrithing,unwindingDN46Ethidiumbromide(溴乙錠)changeDNAsupercoilEthidiumbromide(溴乙錠)changeDN47problemset1.計(jì)算一個(gè)260bps的環(huán)狀DNA在松弛態(tài)和w=-2的L和超螺旋密度。松弛態(tài)：L=T=260/10=26超螺旋：T=26；L=T+W=26+（-2）=24；=-2/24=-0.082.(已知自然界存在大多數(shù)DNA分子的superhelicaldensity（超螺旋密度）是-0.06，一個(gè)10000bpDNA分子，假定是B型構(gòu)像，將形成多少圈超螺旋？3.為什么DNA傾向形成負(fù)超螺旋而不是正超螺旋？problemset1.計(jì)算一個(gè)260bps的環(huán)狀DN48RNARNA49tRNAtRNA504.6TheBiologicalFunctionsofNucleicAcids1、Genome2、Transcription3、Translation:rRNA(ribosome:Rizyme,Structure)mRNAtRNA4.6TheBiologicalFunctionso51分子生物學(xué)中心法則分子生物學(xué)中心法則52英文課件核酸53M1RNAandSRPM1RNA：是E.coli的RNaseI的組成成分，該酶參與轉(zhuǎn)錄的RNA加工。SRP:信號(hào)識(shí)別顆粒M1RNAM1RNAandSRPM1RNA：是E.coli的544.7StabilityofNucleicAcidSecondaryandTertiaryStructureStabilityandDenaturationDenaturation:(1)Theelectrostaticrepulsionofthenegativechargesonthephosphategroups.(2)Thehigherentropyofthedenaturedstate(thedenaturedformhasmorepossibleconformationsthanthedoublehelixform,sothedenaturedformhasgreaterrandomness).Stability:(1)hydrogenbonds(2)ThevanderWaalsinteractionsbetweentheplanarbases4.7StabilityofNucleicAcid55英文課件核酸56Factorsfavoringdissociationofdoublehelicestorandomcoils1、Hypochromism-absorptionoflightbybasesreducedwheninhelix=denaturationcausesincreaseinabsorbanceoflightat260nm.MeltingTemperature(Tm)：Factorsfavoringdissociation57ThefactorsaffectingonTm：①G-C（0.15mol/LNaCl//0.15mol/Lsodiumcitrate）Tm=69.3+0.41（G+C）②DNApurity③pH,ThefactorsaffectingonTm：58問題1、如何保存DNA？A.無水乙醇中；b.純水中；c.75%乙醇中；2、應(yīng)該如何配制DNA溶液，可使其比較穩(wěn)定？A.純水配制；b.1mol/LNaCl配制；c.0.01mol/LNaCl配制；d.0.5mol/LNaOH配制。問題1、如何保存DNA？A.無水乙醇中；b.純水中；c.59溶解度：溶于水酸堿性：生理pH帶負(fù)電荷分子量和形狀：DNA108~1012，d/l=10-7，“柔性棒”——高黏度不對(duì)稱性——正旋光性紫外吸收——260nm有最大光吸收核酸的離心沉降DNA的變性與復(fù)性溶解度：溶于水60思考題1、同一種DNA，變性后以下指標(biāo)可能有什么變化？為什么？黏度、光吸收、沉降系數(shù)、浮力密度2、已知EB（溴乙錠、菲錠溴紅）是一種熒光試劑，如何用EB鑒別質(zhì)粒DNA和雙鏈線形DNA。思考題1、同一種DNA，變性后以下指標(biāo)可能有什么變化？為什么61附：核酸的化學(xué)合成附：核酸的化學(xué)合成62英文課件核酸63DNA限制圖譜和DNA的序列測(cè)定1、DNA的測(cè)定策略DNA限制圖譜和DNA的序列測(cè)定1、DNA的測(cè)定策略64DNA片段的測(cè)定1）Sanger的加減法（1975）與鏈終止法（1977）（chainterminationmethod）步驟：（1）新鏈合成反應(yīng)。反應(yīng)混合物——待測(cè)DNA（順序未知）+寡聚核苷酸引物（順序已知）+dNTPs（四種三磷酸核苷的混合物，其中一種含放射性P）+DNA聚合酶。分四管進(jìn)行。（2）分別加入ddATP、ddCTP、ddGTP、ddTTP做抑制劑。隨機(jī)進(jìn)行鏈終止。（3）聚丙烯酰胺凝膠電泳分離DNA片段（4）放射自顯影、讀序DNA片段的測(cè)定1）Sanger的加減法（1975）與鏈終止65例：待測(cè)DNA3’——ATGCTG——5’（模板）引物5‘—3’——ATGCTG5’——TACGAC5’——TACGA5’——TACG5’——TAC5’——TA5’——T—TACGA—TA—T—TACG—TACGAC—TAC例：待測(cè)DNA—TACGA—T—TACG—TACGAC66根據(jù)Sanger的方法獲得的DNA片段混合物電泳后的放射自顯影圖譜，可以直接在膠板上讀序，方向由下向上。根據(jù)Sanger的方法獲得的DNA片段混合物電泳后的放射自顯672）化學(xué)裂解法（Maxam-GilbertMethod）步驟：（1）單鏈DNA末端標(biāo)記（2）化學(xué)裂解磷酸二酯鍵（3）聚丙烯酰胺凝膠電泳分離DNA片段（4）放射自顯影，讀序2）化學(xué)裂解法（Maxam-GilbertMethod）68英文課件核酸69英文課件核酸70BIOCHEMISTRYNEUCLEICACIDSBIOCHEMISTRYNEUCLEICACIDS71CONTENTS4.1TwoNucleicAcids:DNAandRNA4.2Propertieofnucleotides核苷酸4.3PrimaryStructureofNucleicAcids核酸的一級(jí)結(jié)構(gòu)4.4SecondaryStructureandtertiaryStructureofNucleicAcids核酸的二級(jí)結(jié)構(gòu)和三級(jí)結(jié)構(gòu)4.5StabilityofSecondaryandTertiaryStructure核酸的二級(jí)結(jié)構(gòu)和三級(jí)結(jié)構(gòu)的穩(wěn)定性CONTENTS4.1TwoNucleicAcids:72英文課件核酸734.2NucleotidesNucleotides=Base+Sugar+Phosphate

Nucleoside=Sugar+Base(nophosphate)PRdRPbasebaseRiboseordeoxyribose(核糖或脫氧核糖）4.2NucleotidesNucleotides=74adenine（腺嘌呤）urecil（尿嘧啶）thymine（胸腺嘧啶）cytosine（胞嘧啶）guanine（鳥嘌呤）PurinebasesPyrimidineBasesadenine（腺嘌呤）urecil（尿嘧啶）thymine75StandardNucleotides：5’-adenosinemonophosphate5’-AMP（5’-腺苷酸）5’-guanosinemonophosphate5’-GMP（5’-鳥苷酸）5’-cytidinemonophosphate5’-CMP（5’-胞苷酸）5’-uridinemonophosphate5’-UMP（5’-脲苷酸）5’-deoxyadenosinemonophosphate5’-dAMP（5’-脫氧腺苷酸）5’-deoxyguanosinemonophosphate5’-dGMP（5’-脫氧鳥苷酸）5’-deoxycytidinemonophosphate5’-dCMP（5’-脫氧胞苷酸）5’-deoxythymidinemonophosphate）5’-dTMP（5’-脫氧胸腺苷酸StandardNucleotides：5’-adeno76英文課件核酸77MinorBasesorModifiedbases

(稀有堿基或修飾堿基)MinorBasesorModifiedbases

78Nucleotidepolyphosphate多磷酸核苷8NDPs（5’-nucleotidediphosphate5’-核苷二磷酸）8NTPs（5’-nucleotidetriphosphate5’-核苷三磷酸）ATPNucleotidepolyphosphate多磷酸核苷879CyclicNucleotidesCyclicNucleotides80CoA、NAD+（P）andFADCo-enzymeA（CoA）Nicotinamideadeninedinucleotide（NAD+）煙酰胺腺嘌呤二核苷酸、FlavinAdenineDinucleotide

（FAD）黃素腺嘌呤二核苷酸CoA、NAD+（P）andFADCo-enzyme81NADNAD82NADPNADP83FADFAD84Nucleotidepropertics1.DissociationinwaterThephosphategroupofanucleotideactsasastrongacid(pKa1),Theaminegroupsofthepurineandpyrimidinebasescanbeprotonated.Nucleotidepropertics1.Dissoc852.TautomerizationofthebasesThebasescantautomerize(互變異構(gòu)）;thatis,thebasescanredistributepositionsofhydrogensanddoublebonds2.Tautomerizationofthebases86Unusualbasepair見于酵母苯丙氨酰tRNAUnusualbasepair見于酵母苯丙氨酰tRNA871-methyladenine=T1-methyladenine=T88Nucleotidepropertics3.SpectrumNucleotidesabsorblightstronglyinthenear-ultravioletregionofthespectrum.Nucleotidepropertics3.Spectr89260（X104）AMP1.54GMP1.17CMP0.75dTMP0.92UMP0.99.260（X104）90Biologicalfunctionsofnucleotides1、Themonomericunitsofnucleicacids2、Energycarrier：ATP、GTP、CTP、UTP3、signals：cAMP、cGMP4、coenzymes：FAD、NAD、CoABiologicalfunctionsofnucleo914.3PrimaryStructureofNucleicAcidsTheprimarystructureisthesequenceofnucleosidemonophosphatesDirectionality：5’3’IndividualitypGGTA——,pGpGpTpA——,GGTA——pGGTA（OH）4.3PrimaryStructureofNucle92SignificanceofPrimaryStructure

GeneticinformationstoredinnucleotidesequenceofDNA

GeneisaparticularDNAsequenceSignificanceofPrimaryStruct93

DNAisgeneticsubstance.Avery,MacLeod,McCartyDNAisgeneticsubstance.Aver94HersheyandChase-BacteriophageT2transfersDNAtobacteriaHersheyandChase-Bacterioph954.4SecondarystructureofDNAThesecondarystructurereferstotheshapeanucleicacidassumesasaresultoftheprimarystructure.B-DNA:predominantformintheaqueousenvironmentofthecellA-DNAZ-DNA:purine/pyrimidinetracts4.4SecondarystructureofDN96SecondarystructureofDNA11/0.23nm10/0.34nm12/0.38nmMajorgrooveMinorgrooveSecondarystructureofDNA11/97

98StructureofB-DNA—Thetwochainsinthedoublehelixareantiparallel—Phosphategroupslinktogetherthesugarbackboneviaphosphodiesterbonds.—Thebasesonthetwochainspairinacomplementaryfashion.A=T，G=C—Hydrogenbondsbetweenbasesholdthedoublehelixtogether.(A=T，GC)—B-DNA:d=2nm，pitch=3.4nm,10bp/turnofthehelixStructureofB-DNA—Thetwo99DNAbasepairDNAbasepair100DNAbasepairDNAbasepair101DNAbasepairPrictice:Writethesequence.DNAbasepairPrictice:102ThediscoveryofDNAdoublehelixChargaff'sRule(A=T,G=CinDNA)Franklin,Wilkins:X-rayDiffraction

RefinedStructureThediscoveryofDNAdoublehe103Watson,CrickWatson,Crick104SignificanceofDNAdoublehelixsemi-conservativemodelofDNAreplicationSignificanceofDNAdoublehel105MeselsonandStahlexperiment1958MeselsonandStahlexperiment1106ApplicationofComplementarityDNA半保留復(fù)制核酸分子雜交實(shí)驗(yàn)MoleculehybridizationApplicationofComplementarity107UnusualSecondaryStructuresofDNAPalindromes(回文結(jié)構(gòu)）Cruciformformationinpalindromesequences

GGCGCGCC

CCGCGCGGUnusualSecondaryStructureso108H-DNApurines/pyrimidinesononestrand

H-DNApurines/pyrimidinesonon109英文課件核酸1104.5TertiarystructureofDNATertiarystructurereferstolarge-scalefoldinginalinearpolymerthatisatahigherorderthansecondarystructure.Thetertiarystructureisthespecificthree-dimensionalshapeintowhichanentirechainisfolded.4.5TertiarystructureofDNAT111TertiarystructureofDNATertiarystructureofDNA112CircularDNAandSupercoiling

CircularDNAsSupercoiling:Positive(+)----additionaltwistsaddedbeyondthenormalamountforlinearDNAnegative(-)----reducednumbersoftwistscomparedtolinearDNA.CircularDNAandSupercoiling113SupercoilingTwists(T,纏繞數(shù)):Thetotalnumberoftimesthetwostrandsofthehelixcrossovereachother,excludingwrithing.Twistisameasureofhowtightlythehelixiswound.Writhes(W,扭曲數(shù)):Thenumberoftimesthehelixasawholecrossesoveritself-thatis,thenumberofsuperhelicalturnsthatarepresentLinkingnumber(L,連接數(shù)):ThetotalnumberoftimestwostrandsofaDNAhelixcrosseachotherbymeansofeithertwistorwritheL=T+WThesuperhelicaldensity:L/L0,whereL0isthelinkingnumberoftheDNAinitsunstrained(relaxedstate).SupercoilingTwists(T,纏繞數(shù)):114英文課件核酸115Besideswrithing,unwindingDNA,cruciformformation(viapalindromes),triplehelixformation(H-DNA),andZ-DNAformation,canallreducesuperhelicaltension,too.Besideswrithing,unwindingDN116Ethidiumbromide(溴乙錠)changeDNAsupercoilEthidiumbromide(溴乙錠)changeDN117problemset1.計(jì)算一個(gè)260bps的環(huán)狀DNA在松弛態(tài)和w=-2的L和超螺旋密度。松弛態(tài)：L=T=260/10=26超螺旋：T=26；L=T+W=26+（-2）=24；=-2/24=-0.082.(已知自然界存在大多數(shù)DNA分子的superhelicaldensity（超螺旋密度）是-0.06，一個(gè)10000bpDNA分子，假定是B型構(gòu)像，將形成多少圈超螺旋？3.為什么DNA傾向形成負(fù)超螺旋而不是正超螺旋？problemset1.計(jì)算一個(gè)260bps的環(huán)狀DN118RNARNA119tRNAtRNA1204.6TheBiologicalFunctionsofNucleicAcids1、Genome2、Transcription3、Translation:rRNA(ribosome:Rizyme,Structure)mRNAtRNA4.6TheBiologicalFunctionso121分子生物學(xué)中心法則分子生物學(xué)中心法則122英文課件核酸123M1RNAandSRPM1RNA：是E.coli的RNaseI的組成成分，該酶參與轉(zhuǎn)錄的RNA加工。SRP:信號(hào)識(shí)別顆粒M1RNAM1RNAandSRPM1RNA：是E.coli的1244.7StabilityofNucleicAcidSecondaryandTertiaryStructureStabilityandDenaturationDenaturation:(1)Theelectrostaticrepulsionofthenegativechargesonthephosphategroups.(2)Thehigherentropyofthedenaturedstate(thedenaturedformhasmorepossibleconformationsthanthedoublehelixform,