基因克隆与亚克隆.ppt

专题：基因克隆与亚克隆 To insert a fragment of DNA into a vector is a relatively simple process: Restriction enzyme linearize the plasmid. A restriction enzyme cleaved a target DNA to generate potential insert DNAs,vector DNA has been cut with the same enzyme. DNA ligase to link the compatible ends of the two DNAs. * * * DNA cloning makes molecular biology possible Molecular biology seeks to explain the relationships between the structure and function of biological molecules (DNA, RNA, protein, other small regulators) and how these relationships contribute to the operation and control of biochemical processes (replication, transcription, translation) Molecular analysis of proteins, RNA and DNA, and/or their interaction is essential for molecular biology Getting enough large biomolecules for molecular analysis is impossible by direct purification or isolation from the cell directly DNA cloning make it possible DNA cloning is to place a relatively short fragment of a genome, which might contain the gene or other sequence of interest, in an autonomously replicating piece of DNA, known as a vector, forming RECOMBINANT DNA, which can be replicates independently of the original genome, and normally in other host species. Propagation of the host organism containing the recombinant DNA forms a set of genetically identical organism, or a ClONE. This process is called DNA cloning. What is DNA cloning? Prepare interested DNA fragment from a genome (insert) Plasmid preparation (vector) Restriction digestion (trimming the DNA ends) Ligation (join the insert and the vector) Transformation selection (introduce the plasmids into host cells) Assay of the recombinant DNA: (1) is it a right clone? G4 (2) Analyze the biological function of the cloned gene/DNA DNA Cloning: a simplified flow chart G2 G3 G4 G4 H I PCR or Genomic DNA 1973 - Boyer, Cohen Chang Transform E. coli with recombinant plasmid Stanley Cohen Annie Chan Herbert Boyer Kanamycin resistance gene Plasmid