基因操作原理与方法.pptxVIP

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 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 DNA molecules together. 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DNA的鉴定六、基因组与RNA的提取七、文库构建八、核酸探针九、DNA序列测定十、基因定点突变十一、基因表达基因工程的基本过程目的基因的获得载体的酶切目的基因与载体的连接连接物转化大肠杆菌转化子的筛选重组质粒的鉴定工具酶及其应用 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。 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。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 cells 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 ty