基因操作原理与方法
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基因操作原理与方法
基因操作常用技术
一、宿主与质粒、感受态的制备 二、重组质粒的提取 三、基因工程载体 四、DNA的酶切 五、DNA片段的回收 六、 DNA片段与载体的连接 四、基因克隆策略 五、重组DN十、基因定点突变 十一、基因表达
基因操作原理与方法
Restriction endonucleases
are found in a wide range of bacterial species. Werner Arber discovered that their biological function is to recognize and cleave foreign DNA (e.g., the DNA of an infecting virus); such DNA is said to be restricted. The cell's own DNA is not cleaved because the sequence recognized by the restriction endonuclease is methylated (and thereby protected) by a specific DNA methylase. The restriction endonuclease and the corresponding methylase in a bacterium are sometimes referred to as a restriction-modification system. There are three types of restriction endonucleases, designated I, II, and III. Types I and III are generally large, multisubunit complexes containing both the endonuclease and methylase activities. Type I restriction endonucleases cleave DNA at random sites that can be 1,000 base pairs or more from the recognition sequence. Type III enzymes cleave the DNA about 25 base pairs from the recognition sequence. The type II restriction enzymes, first isolated by Hamilton Smith, are simpler, require no ATP, and cleave the DNA within the recognition sequence itself?The extraordinary utility of the type II enzymes was first demonstrated by Daniel Nathans, and these are the enzymes used most widely for recombinant DNA work.
DNA molecules
together.
基因操作原理与方法
基因操作原理与方法
基因操作原理与方法
基因工程(Gene engineering)是指在体外按既 定的目的和方案，将一目的基因片段，与载体 结合，构成DNA重组体，并将其引入受体细胞 中。重组体随细胞繁殖而扩增，同时也可进行 基因表达，产生特定的基因产物或使受体细胞 表现出新的的遗传性状。基因工程又称遗传工 程 (Genetic engineering)，重组DNA (Recombinant DNA)技术，DNA克隆(DNA cloning)或分子克隆(Molecular cloning)技术。
基因操作原理与方法
基因工程的基本过程
1. 目的基因的获得 2. 载体的酶切 3. 目的基因与载体的连接 4. 连接物转化大肠杆菌 5. 转化子的筛选 6. 重组质粒的鉴定
基因操作原理与方法
工具酶及其应用
分子生物学中常用的工具酶有限 制性内切酶、DNA连接酶、Rnase A、 碱性磷酸酶、DNA聚合酶、T4多聚核 苷酸激、核酸酶
基因工程原理与方法
基因操作、基因重组、基因工程 基因克隆、分子克隆
基因操作原理与方法
基因操作原理与方法
First, restriction endonucleases cleave DNA at specific sequences to generate a set of smaller fragments. Second, the DNA fragment to be cloned can be isolated and joined to a suitable cloning vector using DNA ligase to seal the
基因操作原理与方法
一限制性内切酶与连接酶
Restriction endonucleases & ligase 能识别并切割dS-DNA分子内特殊核甘酸序 列的酶称为限制性核酸内切酶。
基因操作原理与方法
细菌的限制与修饰系统
1952午Luria和Human在研究T偶数噬茵体 及1953年Bertani和Weigle在研究λ和P2噬菌体的 宿主范围时发现:当一个噬茵体从其天然宿主E. coli品系A转到另一个品系B细胞中时，往往不 能生长。他们把此现象称为宿主控制性限制现 象 1962年，Arber及其同事们作了大量工作， 经过放射性同位素标记证明，噬菌体在新品系 中的损害伴随有其DNA的降解，但宿主自己的 DNA并不降解，他们提出了限制-修饰酶假说 来解释这种现象。
不同来源、不同物种之间的基因之所以能重组， 并按表现出预计的性状，其原因？
基因操作原理与方法
DNA Cloning: The Basics
To clone means to make identical copies; it is a term that was once restricted to the procedure of isolating one cell from a larger population of cells, then allowing it to reproduce itself to generate many identical cells. In such a way, sufficient quantities of a single cell type were made available for study. By analogy, DNA cloning involves separating a specific gene or segment of DNA from its larger chromosome and attaching it to a small molecule of carrier DNA, then replicating this modified DNA thousands or even millions of times. The result is a selective amplification of that particular gene or DNA segment.
基因操作常用技术
一、宿主与质粒、感受态的制备 二、重组质粒的提取 三、基因工程载体 四、DNA的酶切 五、DNA片段的回收 六、 DNA片段与载体的连接 四、基因克隆策略 五、重组DN十、基因定点突变 十一、基因表达
基因操作原理与方法
Restriction endonucleases
are found in a wide range of bacterial species. Werner Arber discovered that their biological function is to recognize and cleave foreign DNA (e.g., the DNA of an infecting virus); such DNA is said to be restricted. The cell's own DNA is not cleaved because the sequence recognized by the restriction endonuclease is methylated (and thereby protected) by a specific DNA methylase. The restriction endonuclease and the corresponding methylase in a bacterium are sometimes referred to as a restriction-modification system. There are three types of restriction endonucleases, designated I, II, and III. Types I and III are generally large, multisubunit complexes containing both the endonuclease and methylase activities. Type I restriction endonucleases cleave DNA at random sites that can be 1,000 base pairs or more from the recognition sequence. Type III enzymes cleave the DNA about 25 base pairs from the recognition sequence. The type II restriction enzymes, first isolated by Hamilton Smith, are simpler, require no ATP, and cleave the DNA within the recognition sequence itself?The extraordinary utility of the type II enzymes was first demonstrated by Daniel Nathans, and these are the enzymes used most widely for recombinant DNA work.
DNA molecules
together.
基因操作原理与方法
基因操作原理与方法
基因操作原理与方法
基因工程(Gene engineering)是指在体外按既 定的目的和方案，将一目的基因片段，与载体 结合，构成DNA重组体，并将其引入受体细胞 中。重组体随细胞繁殖而扩增，同时也可进行 基因表达，产生特定的基因产物或使受体细胞 表现出新的的遗传性状。基因工程又称遗传工 程 (Genetic engineering)，重组DNA (Recombinant DNA)技术，DNA克隆(DNA cloning)或分子克隆(Molecular cloning)技术。
基因操作原理与方法
基因工程的基本过程
1. 目的基因的获得 2. 载体的酶切 3. 目的基因与载体的连接 4. 连接物转化大肠杆菌 5. 转化子的筛选 6. 重组质粒的鉴定
基因操作原理与方法
工具酶及其应用
分子生物学中常用的工具酶有限 制性内切酶、DNA连接酶、Rnase A、 碱性磷酸酶、DNA聚合酶、T4多聚核 苷酸激、核酸酶
基因工程原理与方法
基因操作、基因重组、基因工程 基因克隆、分子克隆
基因操作原理与方法
基因操作原理与方法
First, restriction endonucleases cleave DNA at specific sequences to generate a set of smaller fragments. Second, the DNA fragment to be cloned can be isolated and joined to a suitable cloning vector using DNA ligase to seal the
基因操作原理与方法
一限制性内切酶与连接酶
Restriction endonucleases & ligase 能识别并切割dS-DNA分子内特殊核甘酸序 列的酶称为限制性核酸内切酶。
基因操作原理与方法
细菌的限制与修饰系统
1952午Luria和Human在研究T偶数噬茵体 及1953年Bertani和Weigle在研究λ和P2噬菌体的 宿主范围时发现:当一个噬茵体从其天然宿主E. coli品系A转到另一个品系B细胞中时，往往不 能生长。他们把此现象称为宿主控制性限制现 象 1962年，Arber及其同事们作了大量工作， 经过放射性同位素标记证明，噬菌体在新品系 中的损害伴随有其DNA的降解，但宿主自己的 DNA并不降解，他们提出了限制-修饰酶假说 来解释这种现象。
不同来源、不同物种之间的基因之所以能重组， 并按表现出预计的性状，其原因？
基因操作原理与方法
DNA Cloning: The Basics
To clone means to make identical copies; it is a term that was once restricted to the procedure of isolating one cell from a larger population of cells, then allowing it to reproduce itself to generate many identical cells. In such a way, sufficient quantities of a single cell type were made available for study. By analogy, DNA cloning involves separating a specific gene or segment of DNA from its larger chromosome and attaching it to a small molecule of carrier DNA, then replicating this modified DNA thousands or even millions of times. The result is a selective amplification of that particular gene or DNA segment.