[理学]Methods in Molecular biology-chapter 4.ppt

Methods in Molecular biology QiuZheng 邱郑 qiuzheng754@ Charpter 4 Molecular Cloning Methods Why to clone genes? How to clone genes in bacteria and in eukaryotes? 4.1 Gene Cloning A clone: a group of identical cells or organisms. Identical twins constitute a natural clone. The usual procedure in a gene cloning experiment is to place a foreign gene into bacterial cells, isolate individual cells, and grow colonies from each of them. Stanley Cohen, Herbert Boyer, and their colleagues performed the first cloning experiment in 1973. The role of Restriction Endonuclease What about newly replicated DNA? methylated—hemimethylation—methylated Restriction endonucleases cleave DNA molecules at particular sites To name a restriction endonuclease: e.g. Hind II EcoRⅠ Escherichia coli R Genus：Escherichia Species：coli Strain：R Number：Ⅰ Restriction endonucleases: the nucleases that cleave DNA at particular sites by the recognition of specific sequences Sequences with twofold symmetry are called palindromes. (回文结构). e.g. EcoRI Different enzymes recognize their specific target sites with different frequency EcoRI Recognize hexameric sequence: 46 Sau3A1 Recognize terameric sequence: 44 Thus Sau3A1 cuts the same DNA molecule more frequently Enzymes that recognize identical sequences are called isoschizomers, although the cutting sits within these sequences are different. The first cloning experiment involving a recombinant DNA assembled in vitro. Summary Restriction endonucleases recognize specific sequence in DNA molecules and make cuts in both strands. Because the cuts in the two strands are frequently staggered, restriction enzymes can create sticky ends that help link together two DNAs to form a recombinant DNA in vitro. Vectors Vectors: Carriers to allow replication of recombinant DNAs The foreign DNA has no origin of replication, so it can not replicate unless it is placed in a vector that does have an origin of replication. autonom