ChapterManipulationofPurifiedDNA重点分析.ppt

Chapter 4 Manipulation of Purified DNA Contents 4.1 The range of DNA manipulative enzymes 4.1.1 Nucleases 4.1.2 Ligases 4.1.3 Polymerases 4.1.4 DNA modifying enzymes 4.1.5 Topoisomerases Contents 4.2 Enzymes for cutting DNA-restriction endonucleases 4.2.1 The discovery and function of restriction endonucleases 4.2.2 Type II restriction endonucleases cut DNA at specific nucleotide sequences 4.2.3 Blunt ends and sticky ends 4.2.4 The frequency of recognition sequences in a DNA molecule 4.2.5 Performing a restriction digest in the laboratory 4.2.6 Analysing the result of restriction endonuclease cleavage 4.2.7 Estimation of the sizes of DNA molecules 4.2.8 Mapping the positions of different restriction sites on a DNA molecule Contents 4.3 Ligation-joining DNA molecules together 4.3.1 The mode of action of DNA ligase 4.3.2 Sticky ends increase the efficiency of ligation 4.3.3 Putting sticky ends onto a blunt- ended molecule 4.1 The range of DNA manipulative enzymes Five classes based on the reaction types: (1).Nucleases (核酸酶):enzymes that cut, shorten or degrade nucleic acid molecules. (2).Ligases(连接酶): join nucleic acid molecules together. (3).Polymerases (聚合酶)make copies of molecules. (4).Modifying enzymes(修饰酶) remove or add chemical groups. (5).Topoisomerases(拓扑异构酶) introduce or remove supercoils from covalently closed-circular DNA. 4.1 Nuclease(核酸酶) Nucleases degrade DNA molecules by breaking the phosphodiester bonds(磷酸二酯键) that link one nucleotide to the next in a DNA strand. Exonucleases remove nucleotides one at a time from the end of a DNA molecule. Endonucleases :break internal phosphodiester bonds within a DNA molecule. Fig.4.1 The reactions catalysed by the two different kinds of nuclease.