作用原理识别双链DNA未甲基化修饰的特异序列.PPT

11-17-2004 Molecular Biology Chapter 6: Tools for DNA Cloning Host cells Enzymes (restriction and modification enzymes) Vectors References Chapter 15 in : “Essentials of Molecular Biology” Chapter 4, 6, 8 in “Introduction to Genetic Engineering” Why Clone DNA? Isolation and propagation of genes of interest for identification cDNA and genomic libraries construction Preparation for specific probes and DNA sequences Functional expression and/or purification of large amounts of protein In vitro modification of genes of interest Requirement for DNA Cloning Techniques for DNA isolation Enzymes used for DNA manipulation Vectors for carrying foreign DNA Bacterial host cells for storage, amplification and expression of recombinant DNA Technological Breakthrough inMolecular Biology Leading to Cloning Discovery of restriction enzymes and modification enzymes, allowing cutting and manipulation of DNA molecules Methods of isolating DNA molecules and agarose gel electrophoresis for separating DNA molecules Plasmids and bacteriophage as vectors for in vivo amplification of DNA molecules Methods of introducing DNA molecules into cells, “Transformation” Hybridization method for identifying specific DNA molecules I. Host cells How to choose host cells? Depends on what do you plan to do with the cells For cloning or for expression For Cloning: E.coli An ideal host cell Easy to handle and propagate 繁殖 Available as a wide variety of genetically defined strains Accept a range of vectors For Expression Recombinant Protein Expression Depends on: Design of the experiment Promoter specificity on the vector To choose: Prokaryotic Cells Eukaryotic Cells Yeast Insect Mammalian Transgenic, include plant cells II. Enzymes Restriction enzymes Modification enzymes DNA ligase 1. Restriction enzymes Introduction of Restriction Enzymes Types of Restriction Enzymes Properties of Restriction Enzymes Application of Restriction Digestion 3. Properties of Restriction Enzymes Each enzyme recogn