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(xué)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與遺傳工程課件6互補(bǔ)DNA的製造互補(bǔ)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與遺傳工程課件10DNAvector載體：cloningvector選殖載體（Bacterialplasmids細(xì)菌質(zhì)體、Bacteriophages噬菌體）Shuttlevectors穿梭載體（應(yīng)用在酵母菌yeast和細(xì)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與遺傳工程課件14重組DNA與遺傳工程課件15細(xì)菌間質(zhì)體基因的轉(zhuǎn)移細(xì)菌間質(zhì)體基因的轉(zhuǎn)移16Hostorganisms宿主:(1)

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

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

Animalorplantcell:優(yōu)點(diǎn)：高等生物細(xì)胞的表現(xiàn)，缺點(diǎn)：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與遺傳工程課件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(zhǎng)基因的老鼠轉(zhuǎn)殖生長(zhǎng)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(xì)菌轉(zhuǎn)殖植物細(xì)胞的方法利用細(xì)菌轉(zhuǎn)殖植物細(xì)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與遺傳工程課件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與遺傳工程課件40DNA自動(dòng)定序儀DNA自動(dòng)定序儀41DNA自動(dòng)定序儀DNA自動(dòng)定序儀42autosequencerautosequencer43脂蛋白與膽固醇HDLs:高密度脂蛋白 將膽固醇運(yùn)送到肝臟加以代謝LDLs:低密度脂蛋白 將膽固醇運(yùn)送到利用或儲(chǔ)存細(xì)胞膽固醇所引起的冠狀動(dòng)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(dòng)脈44Vaccines疫苗 Proteincoatofvirusorbacteria. Modifypathogen'sgenomeVaccines疫苗 Proteincoatofvi45Pharmaceuticalproducts

藥品製造 Insulin胰島素

Growthhormones生長(zhǎng)激素

EPO(erythropoietin)紅血球生長(zhǎng)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與遺傳工程課件49DNA指紋辨識(shí)DNA指紋辨識(shí)50Environment環(huán)保DetoxificationminingEnvironment環(huán)保Detoxification51請(qǐng)舉例說明運(yùn)用在生物科技中的物理、化學(xué)基本技術(shù)和原理請(qǐng)舉例說明運(yùn)用在生物科技中的物理、化學(xué)基本技術(shù)和原理52參考習(xí)題：怎樣從細(xì)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(biāo)？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(qǐng)?zhí)嵋粋€(gè)可能解決現(xiàn)代環(huán)境問題的遺傳工程技術(shù)。參考習(xí)題：怎樣從細(xì)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(xué)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與遺傳工程課件59互補(bǔ)DNA的製造互補(bǔ)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與遺傳工程課件63DNAvector載體：cloningvector選殖載體（Bacterialplasmids細(xì)菌質(zhì)體、Bacteriophages噬菌體）Shuttlevectors穿梭載體（應(yīng)用在酵母菌yeast和細(xì)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與遺傳工程課件67重組DNA與遺傳工程課件68細(xì)菌間質(zhì)體基因的轉(zhuǎn)移細(xì)菌間質(zhì)體基因的轉(zhuǎn)移69Hostorganisms宿主:(1)

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

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

Animalorplantcell:優(yōu)點(diǎn)：高等生物細(xì)胞的表現(xiàn)，缺點(diǎn)：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與遺傳工程課件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(zhǎng)基因的老鼠轉(zhuǎn)殖生長(zhǎng)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(xì)菌轉(zhuǎn)殖植物細(xì)胞的方法利用細(xì)菌轉(zhuǎn)殖植物細(xì)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與遺傳工程課件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)