Genetic Recombination and Genetic Engineering DNA DNA Recombination

第十四章基因重组和基因程 Genetic Recombination and Genetic Engineering 目录

第一节 DNA的重组 DNA Recombination

DNA重组同源重组 (homologous recombination) 接合作用 (conjugation) 转化作用 (transformation) 转导作用 (transduction) 位点特异的重组(site-specific recombination) 转座 (transposition)

（二）转化作用通过自动获取或人为地供给外源DNA，使细胞或培养的受体细胞获得新的遗传表型，称为转化作用 (transformation)。 Transformation: Introduction of an exogenous DNA into a cell, causing the cell to acquire a new phenotype.

四、转座重组由插入序列和转座子介导的基因移位或重排称为转座(transposition)。 Transposition: The movement of a gene or set of genes from one site in the genome to another.

（一）插入序列转座插入序列(insertion sequences, IS)组成：二个分离的反向重复(inverted repeats, IR)序列特有的正向重复序列一个转座酶（transposase)编码基因 IR Transposase Gene IR 发生形式：保守性转座(conservative transposition) 复制性转座(duplicative transposition)

（二）转座子转座转座子(transposons) ——可从一个染色体位点转移到另一位点的分散重复序列。 Transposon (transposable element): A segment of DNA that can move from one position in the genome to another. 转座子组成：反向重复序列转座酶编码基因抗生素抗性等有用的基因 IR Transposase Gene 有用基因 IR

v Recombinant DNA technology has revolutionized the study of the gene. It is a newly technology based on well understanding theories of gene transfer and recombination. There are three broad classes of recombination events. 1) Homologous recombination is recombination between two DNA molecules of similar sequence, occurring in all cells; occurs during meiosis and mitosis in eukaryotes. 2) Site -specific recombination is a type of genetic recombination that occurs only at specific sequences depending on an enzyme-mediated recognition. 3) Transposition recombination is the movement of a gene or a set of genes from one site in the genome to another.

v The bacterial gene transfer includes conjugation, transformation and transduction. Conjugation is DNA plasmid transfers from cell to cell by conjunct between cells or flagellum conjunct between bacteria. Transformation is the introduction of an exogenous DNA into a cell, which will acquire a new phenotype. Transduction is the transfer of genetic information from one cell to another by means of a viral vector. Covalent link between different DNA molecular is recombined in the course of conjugation, transformation, transduction and transposition.

第二节重组DNA技术 DNA Recombination Technique

一、重组DNA技术相关概念（一） DNA克隆克隆(clone): 来自同一始祖的相同副本或拷贝的集合。 Clone: A population of identical cells or DNA molecules descended from a single progenitor. Also viruses or organisms that are genetically identical and descended from a single progenitor. 获取同一拷贝的过程称为克隆化(cloning)，即无性繁殖。

v DNA cloning: Recombinant DNA technique in which specific c. DNAs or fragments of genomic DNA are inserted into a cloning vector, which then is incorporated into cultured host cells (e. g. , E. coli cells) and maintained during growth of the host cells; also called gene cloning.

v Particularly important to Genetic engineering is a set of enzymes made available by decades of research on nucleic acid metaboism. Two enzymes in particular lie at the heart of the general approach to generating and propagating a recombinant DNA molecule, restriction endonucleases and DNA ligase

v Restriction enzyme (endonuclease): Any enzyme that recognizes and cleaves a specific short sequence, the restriction site, in doublestranded DNA molecules. These enzymes are widespread in bacteria and are used extensively in recombinant DNA technology.

HindⅡ GTCGAC CAGCTG GTC GAC CAG + CTG 平端切口 Bam HⅠ GGATCC CCTAGG G + GATCC G CCTAG 粘端切口

（三）目的基因 v c. DNA (complementary DNA) v 基因组DNA (genomic DNA)

克隆载体(cloning vector) 为使插入的外源DNA序列被扩增而特意设计的载体称为克隆载体。 Cloning vector: An autonomously replicating genetic element used to carry a c. DNA or fragment of genomic DNA into a host cell for the purpose of gene cloning. Commonly used vectors are bacterial plasmids and modified bacteriophage genomes.

vector) 为使插入的外源DNA序列可转录翻译成多肽链而特意设计的载体称为表达载体。 Expression vector: A modified plasmid or virus that carries a gene or c. DNA into a suitable host cell and there directs synthesis of the encoded protein. Some expression vectors are designed for screening DNA libraries for a gene of interest; others, for producing large amounts of a protein from its cloned gene. v 表达载体(expression

v Plasmid: Small, circular extrachromosomal DNA molecule capable of autonomous replication in a cell. Commonly used as a cloning vector.

Cloning a segment of DNA, either prokaryotic or eukaryotic, entails five general procedures v v v l. A method for cutting DNA at precise locations. The discovery of sequence-specific endonucleases (restriction endonucleases) provided the necessary molecular scissors. 2. A method for joining two DNA fragments covalently. DNA ligase can do this. 3. Selection of a small molecule of DNA capable of self replication. Segments of DNA to be cloned can be joined to plasmids or viral DNAs (cloning vectors). These composite DNA molecules containing covalently linked segments derived from two or more sources are called recombinant DNAs. 4. A method for moving recombinant DNA from the test tube into a host cell that can provide the enzymatic machinery for DNA replication. 5. Methods to select or identify those host cells that contain recombinant DNA.

v Chemical synthesis of oligonucleotides by sequential addition of reactive nucleotide derivatives in the 3 → 5 direction. The first nucleotide (monomer 1) is bound to a glass support by its 3 hydroxyl; its 5 hydroxyl is available for addition of the second nucleotide. The second nucleotide in the sequence (monomer 2) is derivatized by addition of 4 , 4 -dimethoxytrityl (DMT) to its 5 hydroxyl, thus blocking this hydroxyl from reacting; in addition, a highly reactive methylated diisopropyl phosphoramidite group (red letters) is attached to the 3 hydroxyl. When the two monomers are mixed in the presence of a weak acid, they form a 5 → 3 phosphodiester bond with the phosphorus in the trivalent state. Oxidation of this intermediate with iodine (I 2) increases the phosphorus valency to 5, and subsequent removal of the DMT group by detritylation with zinc bromide (Zn. Br 2) frees the 5 hydroxyl. Monomer 3 then is added, and the reactions are repeated. Repetition of this process eventually yields the entire oligonucleotide. Finally, all the methyl groups on the phosphates are removed at the same time at alkaline p. H, and the bond linking monomer 1 to the glass support is cleaved.

v DNA library: Collection of cloned DNA molecules consisting of fragments of the entire genome (genomic library) or of DNA copies of all the m. RNAs produced by a cell type (c. DNA library) inserted into a suitable cloning vector.

逆向转录制备双链c. DNA Oligo(dt)n m. RNA c. DNA SS-c. DNA DS-c. DNA (含Loop) DS-c. DNA

v v v c. DNA (complementary DNA) DNA molecule copied from an m. RNA molecule by reverse transcriptase and therefore lacking the introns present in genomic DNA. Sequencing of a c. DNA permits the amino acid sequence of the encoded protein to be deduced; expression of c. DNAs in recombinant cells can be used to produce large quantities of their encoded proteins in vitro. genomic DNA All the DNA sequences composing the genome of a cell or organism. DNA library Collection of cloned DNA molecules consisting of fragments of the entire genome (genomic library) or of DNA copies of all the m. RNAs produced by a cell type (c. DNA library) inserted into a suitable cloning vector.

人接头及其应用 Eco RⅠ 5´- CCGAATTCG 3´- GGCTTAAGC 目录

Southern印迹目录

v The Southern blot technique for detecting the presence of specific DNA sequences following gel electrophoresis of a complex mixture of restriction fragments. The diagram depicts three restriction fragments in the gel, but the procedure can be applied to a mixture of millions of DNA fragments. A similar procedure, called Northern blotting, is used to detect specific RNA sequences.

v Heterogenous DNA fragment can not be propagated autonomously, only by connecting with replicon forming chimera DNA can it be propagated in host cells. The replicon was called vector which also contain a selectable genetic element such as antibiotic resistance to facilitate the identification of cells harboring the recombinant vector. The vectors can be reconstructed from bacterial plasmids, phages and viruses DNA. After introduction the recombinant plasmids into the recipient bacteria, the bacteria containing plasmid can grow on agar plate containing specific antibiotics. Screening and identifying those antidrugs bacteria further can obtain clones containing target gene by in situ hybridization, southern blot and immunological techniques, and then amplifying and separating recombinant DNA to get target gene clones.

v The study of gene structure and function has been greatly facilitated by recombinant DNA technology. The isolation and cloning of a target gene from a gene library or c. DNA library includes acquiring target gene, selecting and reconstructing cloning vector, inserting target gene into vector, introducing the vector with its target gene into a cell in which it can be propagated to form clones, Screening and identifying colonies carrying recombinant plasmid. Bacterial restriction endonucleases and DNA ligases provide the most important instruments for cutting DNA at specific sequences and joining DNA fragments in the course of gene cloning. The target gene can be obtained by chemosynthesis, enzymatic synthesis and PCR amplification.