分子克慢隆工具酶.pptVIP

基因工程 田长恩 Tel:3.2 DNA连接酶（ligase） Nucleases （核酸酶）: cut, shorten or degrade nucleic acid, such as Bal31, ExonucleaseIII, DNaseI, nucleaseS1. Polymerase（聚合酶）: make copies of nucleic acid, such as DNA pol.I, RTase, RNA pol. Modifying enzymes（修饰酶）: remove or add chemical groups, such as BAP/CIP, polynucleotide kinase, terminal trasferase Topoisomerases（拓扑异构酶）: introduce or remove supercoils from covalently closed circular DNA (cccDNA) 5’-3’ Exonuclease Activity 3’-5’ Exonuclease Activity DNA Polymerase Activity 1） Alkaline Phosphatase （碱性磷酸酶）: BAP/CIP 1 名词解释 限制性核酸内切酶；同裂酶；同尾酶；限制性核酸内切酶星活性；限制性图谱；平末端；粘性末端；衔接头；连接子 2 简要回答下列问题 1）基因工程中常用的酶有哪些？各有何的用途？ 2）何谓定向克隆？如何进行？ 3）说明限制性核酸内切酶产生Star活性的原因及其克服措施。 3.2.1 连接酶的功能  1）缺刻修复 2）连接两个DNA分子 We usually use T4 ligase in joining DNA fragments including Sticky and blunt ends. Mechanism: ligation takes place between 5’-P and 3’-OH 3.2.2 Cloning strategy 3.2.2.1 Ligating blunt ends It is very difficult to join sticky ends of DNA. Under the higher temperature (22℃) condition, large amount of substrates and enzyme should be used to increase the chances of the ends of the molecules coming together in the correct way. 3.2.2.2 ligating sticky ends It is very easily to join sticky ends of DNA because of hydrogen bunds formation between base pairs, the temperature can be at 16℃. So we should try to get sticky ends in gene cloning. 3.2.2.3 Other ways to obtain sticky ends Add linkers(连接体) to blunt ends Linker: A short, sequence-known, synthetic ds-DNA with restriction site and blunt ends 2) Add adaptors （衔接头 ） to blunt ends adaptor: A short, sequence-known, synthetic oligaonucleotides （寡核苷酸） with sticky ends in which restriction site exists. 3.2.2.4 Directional cloning (定向克隆) It is very important to keep correct direction between the insert and linear vector, as the cloned gene must be placed downstream of promoter in correct direction. Promoter Ter