食管癌細(xì)胞ngal基因端轉(zhuǎn)錄調(diào)控區(qū)不同長度片段pcr擴增

1、section 1DNA Recombinationsection 2Recombinant DNA technologysection 3Relationship between Recombinant DNA technology and Medicine,section 1DNA Recombination,1.1 Homologous Recombination1.2 Gene Transfer and Reco

2、mbine in Bacteria Conjugation Transformation Transduction1.3 Site-specific Recombination1.4 Transpositional Recombination,1.1 Homologous Recombination The covalence connection between different DNA mol

3、eculars is called DNA recombination or gene recombination The gene recombination includes two types as follows ? homologous recombination ? site-specific recombination ? transpositional recombi

4、nation,Transformation? There is foreign DNA. ? The phenotype of organisms is changed. ? The changed Phenotype is passed down stably,Transduction? When virus is released from infected one cell and go to i

5、nfect other cell, the DNA fragment transfer from one cell to other cell. This is called the transduction.,? DNA fragment transformation between two bacterium? phage is carrier,1.4 Transpositional recombi

6、nationthe displacement of some gene in the genome by insertion sequence or transposons,1.4.1 insertion sequence and its mediated gene transposition? The length of typical insertion sequence is about

7、750~1500bp.? Typical insertion sequence includes two 9 ~ 41bp inverted repeat sequence and a transposase.? A 4 ~12bp positive repeat sequence always link to flanking of inverted repeat sequence. ? Gene t

8、ransposition by insertion sequence: ? conservative transposition ? duplication transposition,1.4.2 structure of transposons? The transposon is a dispersive and repeat sequence in the genome. ? The

9、transposon can transfer from one region to other region of the genome.,,? The structure of transposon is similar to one of insertion sequence.? The both insertion sequence and transposon contain transp

10、osase gene and flanking inverted repeat sequences, but transposon also contain a few other genes. ? The insertion sequence is the most simple transposon in fact.,,section 2DNA recombination technology

11、2.1 the basic concept related with DNA recombination technology2.2 the basic principle of DNA recombination technology,2.1 the concept related with DNA recombination technology 2.1.1 DNA cloning 2

12、.1.2 tool enzyme 2.1.3 target gene 2.1.4 gene vector,2.1.1 DNA cloning? It is a process of DNA molecular amplification.? Usually, the first a target DNA fragment is inserted to a vector and a recombinant (r

13、eplicon) is constructed. ? The second the recombinant is transformed into host cell and screen out the cell containing the recombinant.? The last that cell is amplified, namely a mass of target DNA

14、 molecule is gained.,2.1.2 tool enzyme? restriction endonuclease? DNA ligase ? DNA polymerase I? reverse transcriptase? polynucleotide kinase? end-transferase? alkaline phosphatase,2.1.3 target geneThe i

15、nterested gene is the target gene,source of the target gene * It is from genomic DNA directly, this is prokaryotic gene only generally. * It is from artificial synthesis, this is simple polypeptide gene ge

16、nerally. * It is from mRNA. * It is from genomic library or cDNA library. * Polymerase Chain Reaction (PCR).,cDNA library,,transformation,,cDNA library,,,extension,,2.1.4 gene vector ? The gene vectors are DNA

17、 molecules, which structure is reconstructed. ? They can carry target DNA fragment ? The target gene or DNA fragment is amplified and expressed.,vector,2.2 the basic principle of DNA recombination techno

18、logy,,separate target gene,cut and ligate target gene and vector,,,,,,,,,,,target gene expression,,,prokaryotic expression system,D,Expression analysis of four expression vectors in E.coli by SDS-PAGE,,,eukaryotic expre

19、ssion system,,,,,section 3 the relationship between DNA recombination technology and medicine? discover and separate pathogenic gene? biopharmacy? DNA diagnosis? gene therapy? prevent transmissibility d

20、isease,Summary Homologous RecombinationSite-specific RecombinationTransposition Conjugation Transformation TransductionDNA cloning: separate , cut, ligate , transform, screen, ampli

21、fy, express,選擇題練習(xí)基因重組與基因工程,,1. 基因工程的特點是,A 在分子水平上操作,在分子水平上表達(dá)B 在分子水平上操作,在細(xì)胞水平上表達(dá)C 在細(xì)胞水平上操作,在分子水平上表達(dá)D 在細(xì)胞水平上操作,在細(xì)胞水平上表達(dá)E 以上均可以,,2. 限制性核酸內(nèi)切酶不具有哪項特點 ?,A 僅存在于原核細(xì)胞中B 用于重組DNA技術(shù)中的位I類酶C 能識別雙

22、鏈DNA中特定的堿基順序D 具有一定的外切酶活性E 辨認(rèn)得核苷酸序列常具有回文結(jié)構(gòu),,3. 有關(guān)質(zhì)粒的敘述,下列哪項是錯誤的 ?,A 小型環(huán)狀雙鏈DNA分子B 可小到2-3kb, 大到數(shù)百個kbC 能在宿主細(xì)胞中獨立自主地進行復(fù)制D 常含有耐藥基因E 只有一種限制性核酸內(nèi)切酶切口,,4. 下列哪項不是重組DNA的連接方式?,A 粘性末端與粘性末端的連接B 平端與平端的連接C 粘性末端與平端的連接D

23、 人工接頭連接E 同聚物加尾連接,,5. DNA克隆不包括下列哪項步驟?,選擇一個適合的載體限制性核酸內(nèi)切酶在特異位點裂解質(zhì)粒和目的基因用連接酶連接載體DNA與目的DNA,形成重組體用載體的相應(yīng)抗生素抗性篩選含重組體的細(xì)菌重組體用融合法導(dǎo)入細(xì)胞,,6. 下列哪種酶是重組DNA技術(shù)中最重要的?,A 反轉(zhuǎn)錄酶B 堿性磷酸酶C 末端轉(zhuǎn)移酶D DNA聚合酶IE DNA連接酶,,7. 基因工程中通常使用的質(zhì)粒存在于

24、,A 細(xì)菌染色體B 酵母染色體C 細(xì)菌染色體外D 酵母染色體外E 以上均不是,,8. 在已知DNA序列情況下,獲取目的DNA最方便的方法是,A 人工化學(xué)合成B 基因組文庫法C cDNA文庫法D PCR法E 從染色體DNA直接提取,,9. 基因工程中使目的基因與載體拼接的酶是,A DNA聚合酶B RNA聚合酶C DNA連接酶D RNA連接酶E 限制性核酸內(nèi)切酶,,10. 表達(dá)人類蛋白質(zhì)的

25、最理想的細(xì)胞體系是,A E.coli 表達(dá)體系B 原核表達(dá)體系C 酵母表達(dá)體系D 昆蟲表達(dá)體系E 哺乳類細(xì)胞表達(dá)體系,,11. The nucleotide number which restriction enzyme recognize in DNA nucleotide sequence is,A 4, 5 or 6B 5, 6 or 7C 6, 7 or 8D 4, 6 or 8

26、E 4 - 8,,12. The technique used in identification of DNA is,A northern blottingB southern blottingC Western blottingD affinity chromatographyE ion exchange chromatography,,13. The way of gene recombination doe

27、sn’t include,A transformationB transductionC transpositionD change-over轉(zhuǎn)換E integration,,14. The abbreviation of polymerase chain reaction is,A PRCB PERC PDRD BCRE PCR,,,15. 對于重組體的篩選,屬于非直接選擇法的是,A 免疫化學(xué)法

28、B 原位雜交法C southern 印跡D 補救標(biāo)志篩選E 酶聯(lián)免疫篩選,,16. 基因工程中,目的基因地來源有,A 化學(xué)合成B PCR合成C cDNA文庫D 基因組文庫E 組織細(xì)胞中染色體DNA直接提取,,17. 質(zhì)粒DNA等作為基因工程載體必須具備的條件是,A 能獨立自主復(fù)制B 易轉(zhuǎn)化C 易篩選(質(zhì)粒DNA含有抗藥性基因等)D 具有合適的限制性核酸內(nèi)切酶酶切位點E 易提取獲得,,18

29、. 將表達(dá)載體導(dǎo)入真核細(xì)胞的轉(zhuǎn)染方法有,A 磷酸鈣轉(zhuǎn)染B DEAE葡萄糖介導(dǎo)轉(zhuǎn)染C 電穿孔D 脂質(zhì)體轉(zhuǎn)染E 顯微注射,,,19. gene cloning also be called,A DNA recombinationB RNA recombinationC DNA cloningD RNA cloningE protein replication,,20. The enzyme tools