biotechnologie

• Methods for manipulating DNA


  • Restriction enzymes and nucleic acid separation
  • Nucleic acid hybridization
  • Polymerase chain reaction
  • Essentials of molecular cloning
  • Molecular methods for mutagenesis
  • Gene fusions and reporter genes

  
• Genetic engineering


  Using in vitro techniques to modify genetic material in a laboratory
• Restriction enzymes


  • Recognize a specific DNA sequence and cut the DNA at that location
  • More common in prokaryotes than eukaryotes, used as protection against foreign DNA
• Restriction enzymes and nucleic acid separation


  1. Restriction enzyme recognition

  2. Restriction enzyme cleavage

  3. Nucleic acid separation
• Polymerase chain reaction (PCR)


  • Used for amplifying small amounts of DNA
  • Quantitative PCR uses fluorescent probes to monitor the amplification process
• Essentials of molecular cloning


  1. Isolation and incorporation of a piece of DNA into a vector

  2. Replication and manipulation of the cloned DNA

  
• Molecular methods for mutagenesis


  • Cassette mutagenesis
  • Knockout mutations
• Gene disruption


  Occurs when a cassette is inserted into the middle of a gene, causing a knockout mutation
• Gene fusions


  Promoters or coding sequences of genes of interest can be fused with those of reporter genes to study gene regulation

  
• Reporter genes


  Encode proteins that are easy to detect and assay

  
• Gene cloning


  1. Isolation and fragmentation of source DNA

  2. Insertion of DNA fragment into cloning vector

  3. Introduction of cloned DNA into host

  
• Plasmids as cloning vectors


  • Small, easy to isolate DNA
  • Independent origin of replication
  • Multiple copy number
  • Presence of selectable markers
• Ideal host for cloning vectors


  • Capable of rapid growth in inexpensive medium
  • Nonpathogenic
  • Capable of incorporating DNA
  • Genetically stable in culture
• Shuttle vectors


  Vectors that can be stably maintained in two or more unrelated host organisms
• Expression vectors


  Allow for controlled expression of cloned genes