Molecular BiologyXu Liyan.ppt

1、Molecular Biology Xu Liyan,Chapter 14 gene recombination and gene engineering,section 1 DNA Recombination section 2 Recombinant DNA technology section 3 Relationship between Recombinant DNA technology and Medicine,section 1 DNA Recombination,1.1 Homologous Recombination 1.2 Gene Transfer and Recombi

2、ne in Bacteria ConjugationTransformationTransduction 1.3 Site-specific Recombination 1.4 Transpositional Recombination,1.1 Homologous RecombinationThe covalence connection between differentDNA moleculars is called DNA recombinationor gene recombinationThe gene recombination includes two types as fol

3、lows homologous recombination site-specific recombination transpositional recombination,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 infect other cell, the

4、DNA fragment transfer from one cell toother cell. This is called the transduction., DNA fragment transformation between two bacterium phage is carrier,1.4 Transpositional recombinationthe displacement of some gene in the genome by insertion sequence or transposons,1.4.1 insertion sequence and its me

5、diated gene transposition The length of typical insertion sequence is about 7501500bp. Typical insertion sequence includes two 9 41bpinverted repeat sequence and a transposase. A 4 12bp positive repeat sequence always linkto flanking of inverted repeat sequence. Gene transposition by insertion seque

6、nce: conservative transposition duplication transposition,1.4.2 structure of transposons The transposon is a dispersive and repeat sequencein the genome. The transposon can transfer from one region to otherregion of the genome., The structure of transposon is similar to one ofinsertion sequence. The

7、 both insertion sequence and transposon contain transposase gene and flanking inverted repeatsequences, but transposon also contain a few othergenes. The insertion sequence is the most simple transposon in fact.,section 2 DNA recombination technology 2.1 the basic concept related with DNArecombinati

8、on technology 2.2 the basic principle of DNArecombination technology,2.1 the concept related with DNArecombination technology2.1.1 DNA cloning 2.1.2 tool enzyme2.1.3 target gene2.1.4 gene vector,2.1.1 DNA cloning It is a process of DNA molecular amplification. Usually, the first a target DNA fragmen

9、t is inserted to a vector and a recombinant (replicon) 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 molecule is gained.,2.1.2 tool enzyme restriction endonucle

10、ase DNA ligase DNA polymerase I reverse transcriptase polynucleotide kinase end-transferase alkaline phosphatase,2.1.3 target gene The interested gene is the target gene,source of the target gene* It is from genomic DNA directly, this isprokaryotic gene only generally. * It is from artificial synthe

11、sis, this is simplepolypeptide gene generally.* 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 molecules, which structure is reconstructed. They can carry

12、target DNA fragment The target gene or DNA fragment is amplified and expressed.,vector,2.2 the basic principle of DNArecombination technology,separate target gene,cut and ligate target gene and vector,target gene expression,prokaryotic expression system,D,Expression analysis of four expression vecto

13、rs in E.coli by SDS-PAGE,eukaryotic expression system,section 3 the relationship between DNArecombination technology and medicine discover and separate pathogenic gene biopharmacy DNA diagnosis gene therapy prevent transmissibility disease,Summary Homologous Recombination Site-specific Recombination

14、 TranspositionConjugationTransformationTransduction DNA cloning: separate , cut, ligate , transform, screen, amplify, express,选择题练习 基因重组与基因工程,1. 基因工程的特点是,A 在分子水平上操作,在分子水平上表达 B 在分子水平上操作,在细胞水平上表达 C 在细胞水平上操作,在分子水平上表达 D 在细胞水平上操作,在细胞水平上表达 E 以上均可以,2. 限制性核酸内切酶不具有哪项特点 ?,A 仅存在于原核细胞中 B 用于重组DNA技术中的位I类酶 C 能识别双链

15、DNA中特定的碱基顺序 D 具有一定的外切酶活性 E 辨认得核苷酸序列常具有回文结构,3. 有关质粒的叙述,下列哪项是错误的 ?,A 小型环状双链DNA分子 B 可小到2-3kb, 大到数百个kb C 能在宿主细胞中独立自主地进行复制 D 常含有耐药基因 E 只有一种限制性核酸内切酶切口,4. 下列哪项不是重组DNA的连接方式?,A 粘性末端与粘性末端的连接 B 平端与平端的连接 C 粘性末端与平端的连接 D 人工接头连接 E 同聚物加尾连接,5. DNA克隆不包括下列哪项步骤?,选择一个适合的载体 限制性核酸内切酶在特异位点裂解质粒和目的基因 用连接酶连接载体DNA与目的DNA,形成重组体

16、用载体的相应抗生素抗性筛选含重组体的细菌 重组体用融合法导入细胞,6. 下列哪种酶是重组DNA技术中最重要的?,A 反转录酶 B 碱性磷酸酶 C 末端转移酶 D DNA聚合酶I E DNA连接酶,7. 基因工程中通常使用的质粒存在于,A 细菌染色体 B 酵母染色体 C 细菌染色体外 D 酵母染色体外 E 以上均不是,8. 在已知DNA序列情况下,获取目的DNA最方便的方法是,A 人工化学合成 B 基因组文库法 C cDNA文库法 D PCR法 E 从染色体DNA直接提取,9. 基因工程中使目的基因与载体拼接的酶是,A DNA聚合酶 B RNA聚合酶 C DNA连接酶 D RNA连接酶 E 限制

17、性核酸内切酶,10. 表达人类蛋白质的最理想的细胞体系是,A E.coli 表达体系 B 原核表达体系 C 酵母表达体系 D 昆虫表达体系 E 哺乳类细胞表达体系,11. The nucleotide number which restriction enzymerecognize in DNA nucleotide sequence is,A 4, 5 or 6 B 5, 6 or 7 C 6, 7 or 8 D 4, 6 or 8 E 4 - 8,12. The technique used in identification of DNA is,A northern blotting B

18、 southern blotting C Western blotting D affinity chromatography E ion exchange chromatography,13. The way of gene recombination doesnt include,A transformation B transduction C transposition D change-over转换 E integration,14. The abbreviation of polymerase chain reaction is,A PRC B PER C PDR D BCR E

19、PCR,15. 对于重组体的筛选,属于非直接选择法的是,A 免疫化学法 B 原位杂交法 C southern 印迹 D 补救标志筛选 E 酶联免疫筛选,16. 基因工程中,目的基因地来源有,A 化学合成 B PCR合成 C cDNA文库 D 基因组文库 E 组织细胞中染色体DNA直接提取,17. 质粒DNA等作为基因工程载体必须具备的条件是,A 能独立自主复制 B 易转化 C 易筛选(质粒DNA含有抗药性基因等) D 具有合适的限制性核酸内切酶酶切位点 E 易提取获得,18. 将表达载体导入真核细胞的转染方法有,A 磷酸钙转染 B DEAE葡萄糖介导转染 C 电穿孔 D 脂质体转染 E 显微注射,19. gene cloning also be called,A DNA recombination B RNA recombination C DNA cloning D RNA cloning E protein replication,20. The enzyme tools commonly used in gene cloning technique are,A restriction enzyme B DNA polymerase I C DNA ligase D reverse transcriptase E terminal transferase,