1四川大学《基因工程》第一章-工具酶.pdfVIP

第一章 分子克隆工具酶 1 第一节 限制性内切酶 Restriction Enzymes Restriction Enzymes An endonuclease （核酸内切酶）that recognizes specific nucleotide sequences in DNA and breaks the DNA chain at that sites 2 一、限制性内切酶的发现 (The discovery of Restriction Enzymes) 1．细菌限制修饰系统 （Restriction and Modification System of Bacteria) 3  E. coli strain C does not have a functional restriction enzyme /modification system  E. coli K12 makes several restriction enzymes, including Eco RI  Bacterial restriction– modification systems have two components – a restriction endonuclease and a DNA methylase. 4 2．限制性内切酶的发现  1968年，从E.coli K B中分离了I型限制性 内切酶EcoK EcoB  1970年，Smith和Wilox 首次从流感嗜血杆 菌（H. influenzae）Rd菌株中发现并分离到 HindII限制酶  1972年，从E.coli RY13中分离EcoRI  1978年，限制酶发现获得诺贝尔奖 5 限制酶为什么不切割自己的染色体DNA？ The organisms that produce restriction enzymes, also produces enzymes that modify its own DNA so that it will not be cleaved by the restriction enzyme. E. coli RY13: EcoRI, EcoRI methylase 6 The resistance of the hemimethylated DNA site to cleavage protects the bacteria DNA from degradation immediately after semiconservative DNA replication. 7 二、限制性内切酶的分类  I 型 ：识别特定序列，切割位点在距识 别位点至少1000 bp处随机切割 EcoB ：TGA(N) TGCT