重組DNA與遺傳工程課件

TheFlowofGeneticInformationDNARNAProteinTranslationTranscriptionReverseTranscriptionDNAReplicationRNAReplicationDNADNA DNA-dependentDNAPolymerase(DNAReplicase)DNA

RNA DNA-dependentRNAPolymerase(Transcriptase)RNADNA RNA-dependentDNAPolymerase(ReverseTranscriptase)RNA

RNA RNA-dependentRNAPolymerase(RNAReplicase)DNA合成酶、轉(zhuǎn)錄酶、反轉(zhuǎn)錄酶、RNA合成酶TheCentralDogma–分子生物學的中心教條:TheFlowofGeneticInformatio1遺傳工程（重組DNA)遺傳工程（重組DNA)2重組DNA技術(shù)的條件：1.

基本工具：enzymes2.

基因的來源:purifiedDNAorcDNA(complementaryDNA)3.

傳輸工具：transformation,infection,electroporation,microinjection,genegun4.

篩選技術(shù):specificantibodyscreening,hybridization(radioactiveisotopeorfluorescentagentlabel),5.

基因表現(xiàn)：expressionvectors,proteinpurification重組DNA技術(shù)的條件：1.

基本工具：enzyme3ThetoolkitforDNAtechnology:1.

Restrictionenzymes限制脢:DanielNathans,HamiltonSmithandWernerArber(1978NobelPrize)

2.

DNAvector載體

3.Hostorganisms宿主ThetoolkitforDNAtechnology4Restrictionenzymes限制脢:1.DanielNathans,HamiltonSmithandWernerArber(1978NobelPrize),DNAligase黏合脢

Restrictionenzymes限制脢:1.Dani5重組DNA與遺傳工程6互補DNA的製造互補DNA的製造7製備cDNA製備cDNA8分離特定基因的方法分離特定基因的方法9重組DNA與遺傳工程10DNAvector載體：cloningvector選殖載體（Bacterialplasmids細菌質(zhì)體、Bacteriophages噬菌體）Shuttlevectors穿梭載體（應(yīng)用在酵母菌yeast和細菌之上）Retroviruses反轉(zhuǎn)錄病毒（藉由感染的方式）DNAvector載體：cloningvector選11大腸桿菌的質(zhì)體大腸桿菌的質(zhì)體12質(zhì)體的放大圖質(zhì)體的放大圖13重組DNA與遺傳工程14重組DNA與遺傳工程15細菌間質(zhì)體基因的轉(zhuǎn)移細菌間質(zhì)體基因的轉(zhuǎn)移16Hostorganisms宿主:(1)

Bacteria:優(yōu)點：繁殖快速、容易操作，缺點：轉(zhuǎn)錄和轉(zhuǎn)譯調(diào)控機制與真核細胞不同(2)

Yeast:優(yōu)點：真核細胞的調(diào)控，缺點：不易操作(3)

Animalorplantcell:優(yōu)點：高等生物細胞的表現(xiàn)，缺點：不易接納外來DNA

Hostorganisms宿主:(1)

Ba17重組DNA的方法重組DNA的方法18重組DNA與遺傳工程19遺傳工程的應(yīng)用 一、DiagnosisofGeneticDiseases遺傳疾病的診斷 二、HumanGeneTherapy人類基因療法 三、Vaccines疫苗 四、Pharmaceuticalproducts藥品製造

五、Forensic法醫(yī) 六、Environment環(huán)保 七、Agricultural農(nóng)業(yè)遺傳工程的應(yīng)用 一、DiagnosisofGenet20GMO：基因重組生物GMO：基因重組生物21轉(zhuǎn)殖生長基因的老鼠轉(zhuǎn)殖生長基因的老鼠22Agricultural農(nóng)業(yè)Transgenicanimals n

enlargevaluableparts n

resistanttopathagens n

improvedagriculturalproducts,suchaschangeAAscompositionsTransgenicplants Tiplasmid(Tumor-inducingplasmid)

Agrobacteriumtumefaciens根瘤土壤桿菌Antisensegenes反義基因抑制番茄成熟Agricultural農(nóng)業(yè)Transgenicani23利用細菌轉(zhuǎn)殖植物細胞的方法利用細菌轉(zhuǎn)殖植物細胞的方法24轉(zhuǎn)殖致瘤基因的植物轉(zhuǎn)殖致瘤基因的植物25抗除草劑基因的棉花抗除草劑基因的棉花26螢光煙草：轉(zhuǎn)殖作物螢光煙草：轉(zhuǎn)殖作物27HumanGeneTherapy

人類基因療法SCID(severecombinedimmunodeficiency)重度混合免疫不全癥1.retroviralvector+ADA(腺甘脫胺脢)Tlymphocyte

intravenousinjection2.ADAgenebonemarrowcellHumanGeneTherapy

人類基因療法SCI28DiagnosisofGeneticDiseases

遺傳疾病的診斷Cysticfibrosis囊性纖維變性Huntington'sdisease亨氏舞蹈癥

DiagnosisofGeneticDiseases

29重組DNA與遺傳工程30聚合脢連鎖反應(yīng)

-1聚合脢連鎖反應(yīng)

-131聚合脢連鎖反應(yīng)

-2聚合脢連鎖反應(yīng)

-232EnzymePolymerasebindsinglestrandedDNAatapointadjacenttoadouble-strandedstretch,andbindstodNTPs(deoxynucleotidetriphosphates)EnzymePolymerasebindsingles33ComponentsofPCRreaction:Enzyme(Taq,Vent,Pfu)AppropriatebuffersAmpledNTPsTemplateDNAPrimersCofactors(eg.Magnesium)ComponentsofPCRreaction:Enz34Thecycle1Denaturation 94or92℃,30sec/incubation Halfactivityafter30incubationsat94℃for60seceach. 60-120secforthefirststepThecycle1Denaturation35Thecycle2Annealing Tm=4(GCbases)+2(ATbases) 30-60sec/incubationThecycle2Annealing36Thecycle3Extension 72℃,500bp/30sec;500-1500/60secThecycle3Extension37Cycles

Example: Denature Annealing Extension cycles 94 45 72 5 94 55 72 30-35 94 55 72(10min)1

Cycles Example:38ConditionoftemperatureConditionoftemperature39重組DNA與遺傳工程40DNA自動定序儀DNA自動定序儀41DNA自動定序儀DNA自動定序儀42autosequencerautosequencer43脂蛋白與膽固醇HDLs:高密度脂蛋白 將膽固醇運送到肝臟加以代謝LDLs:低密度脂蛋白 將膽固醇運送到利用或儲存細胞膽固醇所引起的冠狀動脈阻塞脂蛋白與膽固醇HDLs:高密度脂蛋白膽固醇所引起的冠狀動脈44Vaccines疫苗 Proteincoatofvirusorbacteria. Modifypathogen'sgenomeVaccines疫苗 Proteincoatofvi45Pharmaceuticalproducts

藥品製造 Insulin胰島素

Growthhormones生長激素

EPO(erythropoietin)紅血球生長素 Interferon（干擾素）Pharmaceuticalproducts

藥品製造 46Forensic法醫(yī)RFLPDNAfingerprintingForensic法醫(yī)RFLP47DNA電泳DNA電泳48重組DNA與遺傳工程49DNA指紋辨識DNA指紋辨識50Environment環(huán)保DetoxificationminingEnvironment環(huán)保Detoxification51請舉例說明運用在生物科技中的物理、化學基本技術(shù)和原理請舉例說明運用在生物科技中的物理、化學基本技術(shù)和原理52參考習題：怎樣從細胞中分離出我們想要研究的基因片段？怎樣鑑定所分離的基因是我們想要找的正確目標？如何在試管中複製DNA？怎樣證明王小姐未來所生的小孩是鄭先生的？請?zhí)嵋粋€可能解決現(xiàn)代環(huán)境問題的遺傳工程技術(shù)。參考習題：怎樣從細胞中分離出我們想要研究的基因片段？53TheFlowofGeneticInformationDNARNAProteinTranslationTranscriptionReverseTranscriptionDNAReplicationRNAReplicationDNADNA DNA-dependentDNAPolymerase(DNAReplicase)DNA

RNA DNA-dependentRNAPolymerase(Transcriptase)RNADNA RNA-dependentDNAPolymerase(ReverseTranscriptase)RNA

RNA RNA-dependentRNAPolymerase(RNAReplicase)DNA合成酶、轉(zhuǎn)錄酶、反轉(zhuǎn)錄酶、RNA合成酶TheCentralDogma–分子生物學的中心教條:TheFlowofGeneticInformatio54遺傳工程（重組DNA)遺傳工程（重組DNA)55重組DNA技術(shù)的條件：1.

基本工具：enzymes2.

基因的來源:purifiedDNAorcDNA(complementaryDNA)3.

傳輸工具：transformation,infection,electroporation,microinjection,genegun4.

篩選技術(shù):specificantibodyscreening,hybridization(radioactiveisotopeorfluorescentagentlabel),5.

基因表現(xiàn)：expressionvectors,proteinpurification重組DNA技術(shù)的條件：1.

基本工具：enzyme56ThetoolkitforDNAtechnology:1.

Restrictionenzymes限制脢:DanielNathans,HamiltonSmithandWernerArber(1978NobelPrize)

2.

DNAvector載體

3.Hostorganisms宿主ThetoolkitforDNAtechnology57Restrictionenzymes限制脢:1.DanielNathans,HamiltonSmithandWernerArber(1978NobelPrize),DNAligase黏合脢

Restrictionenzymes限制脢:1.Dani58重組DNA與遺傳工程59互補DNA的製造互補DNA的製造60製備cDNA製備cDNA61分離特定基因的方法分離特定基因的方法62重組DNA與遺傳工程63DNAvector載體：cloningvector選殖載體（Bacterialplasmids細菌質(zhì)體、Bacteriophages噬菌體）Shuttlevectors穿梭載體（應(yīng)用在酵母菌yeast和細菌之上）Retroviruses反轉(zhuǎn)錄病毒（藉由感染的方式）DNAvector載體：cloningvector選64大腸桿菌的質(zhì)體大腸桿菌的質(zhì)體65質(zhì)體的放大圖質(zhì)體的放大圖66重組DNA與遺傳工程67重組DNA與遺傳工程68細菌間質(zhì)體基因的轉(zhuǎn)移細菌間質(zhì)體基因的轉(zhuǎn)移69Hostorganisms宿主:(1)

Bacteria:優(yōu)點：繁殖快速、容易操作，缺點：轉(zhuǎn)錄和轉(zhuǎn)譯調(diào)控機制與真核細胞不同(2)

Yeast:優(yōu)點：真核細胞的調(diào)控，缺點：不易操作(3)

Animalorplantcell:優(yōu)點：高等生物細胞的表現(xiàn)，缺點：不易接納外來DNA

Hostorganisms宿主:(1)

Ba70重組DNA的方法重組DNA的方法71重組DNA與遺傳工程72遺傳工程的應(yīng)用 一、DiagnosisofGeneticDiseases遺傳疾病的診斷 二、HumanGeneTherapy人類基因療法 三、Vaccines疫苗 四、Pharmaceuticalproducts藥品製造

五、Forensic法醫(yī) 六、Environment環(huán)保 七、Agricultural農(nóng)業(yè)遺傳工程的應(yīng)用 一、DiagnosisofGenet73GMO：基因重組生物GMO：基因重組生物74轉(zhuǎn)殖生長基因的老鼠轉(zhuǎn)殖生長基因的老鼠75Agricultural農(nóng)業(yè)Transgenicanimals n

enlargevaluableparts n

resistanttopathagens n

improvedagriculturalproducts,suchaschangeAAscompositionsTransgenicplants Tiplasmid(Tumor-inducingplasmid)

Agrobacteriumtumefaciens根瘤土壤桿菌Antisensegenes反義基因抑制番茄成熟Agricultural農(nóng)業(yè)Transgenicani76利用細菌轉(zhuǎn)殖植物細胞的方法利用細菌轉(zhuǎn)殖植物細胞的方法77轉(zhuǎn)殖致瘤基因的植物轉(zhuǎn)殖致瘤基因的植物78抗除草劑基因的棉花抗除草劑基因的棉花79螢光煙草：轉(zhuǎn)殖作物螢光煙草：轉(zhuǎn)殖作物80HumanGeneTherapy

人類基因療法SCID(severecombinedimmunodeficiency)重度混合免疫不全癥1.retroviralvector+ADA(腺甘脫胺脢)Tlymphocyte

intravenousinjection2.ADAgenebonemarrowcellHumanGeneTherapy

人類基因療法SCI81DiagnosisofGeneticDiseases

遺傳疾病的診斷Cysticfibrosis囊性纖維變性Huntington'sdisease亨氏舞蹈癥

DiagnosisofGeneticDiseases

82重組DNA與遺傳工程83聚合脢連鎖反應(yīng)

-1聚合脢連鎖反應(yīng)

-184聚合脢連鎖反應(yīng)

-2聚合脢連鎖反應(yīng)

-285EnzymePolymerasebindsinglestrandedDNAatapointadjacenttoadouble-strandedstretch,andbindstodNTPs(deoxynucleotidetriphosphates)EnzymePolymerasebindsingles86ComponentsofPCRreaction:Enzyme(Taq,Vent,Pfu)AppropriatebuffersAmpledNTPsTemplateDNAPrimersCofactors(eg.Magnesium)ComponentsofPCRreaction:Enz87Thecycle1Denaturation 94or92℃,30sec/incubation Halfactivityafter30incubationsat94℃for60seceach. 60-120secforthefirststepThecycle1Denaturation88Thecycle2Annealing Tm=4(GCbases)+2(ATbases) 30-60sec/incubationThecycle2Annealing89Thecycle3Extension 72℃,500bp/30sec;500-1500/60secThecycle3Extension90Cycles

Example: Denature Annealing Extension cycles 94 45 72 5 94 55 72 30-35 94 55 72(10min)