不同的核酸内切限制酶.ppt

最常用的方法 DNA ligase is essentially ‘molecular glue’; with restriction enzymes, it provides the tools for cutting and joining DNA molecules. 大肠杆菌染色体编码的叫做DNA连接酶，由大肠杆菌T4噬菌体DNA编码的叫做T4DNA连接酶。 Although the reactions catalysed by the enzymes of E. coli and T4-infected E. coli are very similar, they differ in their cofactor requirements. The T4 enzyme requires ATP, while the E. coli enzyme requires NAD + . In each case the cofactor is split and forms an enzyme–AMP complex. The complex binds to the nick, which must expose a 5’ phosphate and 3’ OH group, and makes a covalent bond in the phosphodiester chain, as shown in Fig. 3.6 Reverse transcriptase(反转录酶 ) 依赖于RNA的DNA聚合酶，5’ →3’DNA聚合活性，无3’ →5’外切活性 1．Variety： 两种商品化的反转录酶（两个来源） AMV 禽成髓细胞瘤病毒 Mo-MLV(M-MuLV) Moloney鼠白血病病毒 2. Activities： 5’→3’DNA聚合活性（Mg++） RNase H 活性 3．Application： cDNA克隆 5’突出DNA的3’端补平 测序反应 (代替 Klenow 大片段) * * 2.3 Enzymes that modify the ends of DNA molecules Alkaline phosphatase (碱性磷酸酶，去除5’磷酸基团) Function： Removes the phosphate group present at the 5’terminus of a DNA molecule.去除DNA分子5’末端的磷酸基团（若用于载体DNA 5’末端，则可防止DNA片段自身聚合状） * * Polynucleotide kinase (多核苷酸激酶，在5’端增加磷酸基团) organism：From E.coli infected with T4 phage function：Add phosphate groups onto free 5’ terminus.给DNA5’末端增加磷酸基团，形成自由的5’-P，以利于DNA的连接。 terminal deoxynucleotidyltransferase (末端脱氧核苷酸转移酶，末端转移酶) organism： From calf thymus (胸腺) tissue function：Adds one or more deoxyribonucleotides ( 脱氧核糖核苷酸) onto the 3’terminus of DNA molecule.催化脱氧核苷酸添加到DNA分子的3’-OH末端，催化作用不需要模板。 Alkaline phosphatase (碱性磷酸酶) Application of alkaline phosphatase treatment to prevent recircularization of vector plasmid without insertion of foreign DNA. 碱性磷酸酶可使被切开后的载体去磷酸化，防止其自身环化。 * Polynucleotide kinase (多核苷酸激酶) The foreign DNA plus ligated adaptors is phosphorylated at the 5’-termini by polynuleotide kinase. 多核苷酸激酶可使5’末端发生磷酸化，使之能与DNA 3’末端正常连接。 * terminal deoxynucleotidyltransferase(末端脱氧核苷酸转移酶,末端转移酶) organism： From calf thymus (胸腺) tissue （P40） Adds one or m