chapter 15 基因重组与基因工程

nullnullMolecular Biology Xu LiyanChapter 15 gene recombination and gene engineeringnullsection 1 DNA Recombination section 2 Recombinant DNA technology section 3 Relationship between Recombinant DNA technology and Medicinenullsection 1 DNA Recombination1.1 Homologous Recombination 1.2 Gene Transfer and Recombine in Bacteria Conjugation Transformation Transduction 1.3 Site-specific Recombination 1.4 Transpositional Recombinationnull 1.1 Homologous Recombination The covalence connection between different DNA moleculars is called DNA recombination or gene recombination The gene recombination includes two types as follows  homologous recombination  site-specific recombination  transpositional recombinationnullnullnullnullnullTransformation  There is foreign DNA.  The phenotype of organisms is changed.  The changed Phenotype is passed down stably nullnullTransduction  When virus is released from infected one cell and go to infect other cell, the DNA fragment transfer from one cell to other cell. This is called the transduction.nullnull DNA fragment transformation between two bacterium  phage is carriernullnull1.4 Transpositional recombination the displacement of some gene in the genome by insertion sequence or transposons null1.4.1 insertion sequence and its mediated gene transposition  The length of typical insertion sequence is about 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 transposition by insertion sequence:  conservative transposition  duplication transpositionnullnull1.4.2 structure of transposons  The transposon is a dispersive and repeat sequence in the genome.  The transposon can transfer from one region to other region of the genome. null The structure of transposon is similar to one of insertion sequence.  The both insertion sequence and transposon contain transposase gene and flanking inverted repeat sequences, but transposon also contain a few other genes.  The insertion sequence is the most simple transposon in fact.nullnullsection 2 DNA recombination technology 2.1 the basic concept related with DNA recombination technology 2.2 the basic principle of DNA recombination technology null 2.1 the concept related with DNA recombination technology 2.1.1 DNA cloning 2.1.2 tool enzyme 2.1.3 target gene 2.1.4 gene vectornull2.1.1 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 (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.null2.1.2 tool enzyme  restriction endonuclease  DNA ligase  DNA polymerase I  reverse transcriptase  polynucleotide kinase  end-transferase  alkaline phosphatasenullnull2.1.3 target gene The interested gene is the target genenull 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 generally. * It is from mRNA. * It is from genomic library or cDNA library. * Polymerase Chain Reaction (PCR).nullnullcDNA librarytransformationcDNA librarynullextensionnull2.1.4 gene vector  The gene vectors are DNA molecules, which structure is reconstructed.  They can carry target DNA fragment  The target gene or DNA fragment is amplified and expressed. null vectornullnullnull2.2 the basic principle of DNA recombination technologynullnullseparate target gene nullcut and ligate target gene and vectornullnullnullnull null nullnullnullnull target gene expressionprokaryotic expression systemDnullExpression analysis of four expression vectors in E.coli by SDS-PAGE nulleukaryotic expression systemnullnullsection 3 the relationship between DNA recombination technology and medicine  discover and separate pathogenic gene  biopharmacy  DNA diagnosis  gene therapy  prevent transmissibility diseasenullSummary Homologous Recombination Site-specific Recombination Transposition Conjugation Transformation Transduction DNA cloning: separate , cut, ligate , transform, screen, amplify, expressnull选择题练习 基因重组与基因工程null1. 基因工程的特点是A 在分子水平上操作,在分子水平上达 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null2. 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null3. 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null4. 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null5. 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 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null6. 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null7. 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null8. 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null9. 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 限制性核酸内切酶null10. 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null11. The nucleotide number which restriction enzyme recognize 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 - 8null12. The technique used in identification of DNA is A northern blotting B southern blotting C Western blotting D affinity chromatography E ion exchange chromatographynull13. The way of gene recombination doesn’t include A transformation B transduction C transposition D change-over转换 E integrationnull14. The abbreviation of polymerase chain reaction is A PRC B PER C PDR D BCR E PCRnull15. 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null 16. 基因工程中,目的基因的来源有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null 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null 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null19. gene cloning also be called A DNA recombination B RNA recombination C DNA cloning D RNA cloning E protein replicationnull 20. The enzyme tools commonly used in gene cloning technique areA restriction enzyme B DNA polymerase I C DNA ligase D reverse transcriptase E terminal transferasenullnull祝大家好运！