05-基因工程第二章课件.ppt

第二章 基因工程的工具酶 第一节 限制性核酸内切酶 一、限制性核酸内切酶的发现 有关的几个概念 限制 (restriction) 修饰 (modification) 宿主控制性限制(host—controlled restriction) 限制性核酸内切酶的生物学功能 Restriction endonucleases Restriction-modification systems occur in many bacterial species, and constitute a defense mechanism against the introduction of foreign DNA into the cell. They consist of two components; the first is a restriction endonuclease, which recognizes a short, symmetrical DNA sequence, and cuts (hydrolyzes) the DNA backbone in each strand at a specific site within that sequence. Foreign DNA will hence be degraded to relatively short fragments. The second component of the system is a methylase, which adds a methyl group to a C or A base within the same recognition sequences in the cellular DNA. This modification renders the host DNA resistant to degradation by the endonuclease. Restriction endonucleases Restriction endonucleases are bacterial enzymes which cut (hydrolyze) DNA into defined and reproducible fragments. In bacteria, they form part of the restriction-modification defense mechanism against foreign DNA.They are the basic tools of gene cloning. 二、限制性核酸内切酶的分类 核酸酶 核糖核酸酶（RNase） 脱氧核糖核酸酶（DNase） 核酸外切酶（exonuclease） 核酸内切酶（endonuclease） 二、限制性核酸内切酶的分类 1、Ⅰ型限制性核酸内切酶 类型I限制性内切酶属于复合核酸酶，既具有内切酶的活性又具有甲基化酶的活性 Messelson 、Yuan EcoK EcoB: T- G - A - N8 - T- G - C - T 5`---- TGA*NNNNNNNN T GCT---------3` 3`---- ACT MMMMMMM A*CGA---------5` 约1000bp 切割 2、Ⅱ型限制性核酸内切酶 （1）一般性质 一般是37℃ HaeⅢ 70 ℃、 SamⅠ25 ℃、 TaqⅠ65 ℃ 牛血清白蛋白（BSA）和二硫苏糖醇（DTT） 1单位酶活性(1unit 1U)： 星活性(star activity)：EcoR Ⅰ* (2)Ⅱ型酶的识别与切割顺序II型酶的识别序列一般是4-6个碱基，也有6个以上的，但没有4个以下的，这些序列具有双重螺旋对称的结构形式，换言之，这些核苷酸对的顺序是呈回文结构。 EcoR Ⅰ： 5`-------GAATTC------3` 3`-------CTTAAG------5` ①平末端（blunt/flush end/termini) HaeⅢ： 5`----GGCC-----3` 5`----GG CC-----3` 3`----CCGG-----5`