• The scientific revolution
  • 1978 Nobel Prize in Medicine to Werner Arber, Ham Smith, and Daniel Nathans for discovering restriction endonucleases
  • 1980 Nobel Prize in Chemistry to Paul Berg for developing "recombinant DNA" techniques
  • 1980 Nobel Prize in Chemistry to Wally Gilbert and Fred Sanger for developing DNA sequencing techniques
  • 1993 Nobel Prize in Chemistry to Kary Mullis for developing PCR
  • 1993 Nobel Prize in Chemistry to Michael Smith for developing site-directed mutagenesis

• Cloning
  • Restriction endonucleases
    • Function of restriction endonucleases
    • Structure of restriction endonucleases (a link to the Biomolecular Structure and Modeling group site at the University of London, UK)
    • Properties of specific restriction endonucleases
      • Fermentas catalog descriptions
      • New England Biolabs catalog descriptions

  • T4 DNA ligase

  • Cloning vectors
    • Phage vectors
    • Plasmid vectors
    • Alpha-complementation
    • Expression vectors

• Oligonucleotide synthesis

• DNA sequencing
  • Maxam-Gilbert sequencing
  • Dideoxy sequencing

• Polymerase chain reaction (PCR)

• Site-directed mutagenesis
  • Site-directed mutagenesis by incorporation of mutant oligonucleotides
  • PCR mutagenesis
  • Using site-directed mutagenesis to test sequence motifs
    • Structure of amino acids
    • Properties of amino acids
    • Codon usage
      • Codon usage in E. coli
      • Codon usage in other organisms
    • Evolutionary constraints on amino acid substitutions
    • Alanine scanning
    • An example of a site-directed mutagenesis experiment

• Red swaps - targeted linear recombination in bacteria

• DNA Shuffling (aka Sexual PCR) - generation of new variants by reshuffling gene families

• Some sample problems on DNA cloning and sequencing.
• Some sample problems on restriction mapping.
• Some sample problems on PCR and site-directed mutagenesis.

REFERENCES:

• Court D, Sawitzke J, Thomason L. 2002. Genetic engineering using homologous recombination. Annu Rev Genet. 36: 361-88.
• Funnell, B., and G. Phillips. 2004. Plasmid Biology. American Society for Microbiology Press, Washinton DC.
• Poteete A. 2001 What makes the bacteriophage lambda Red system useful for genetic engineering: molecular mechanism and biological function. FEMS Microbiol Lett. 201(1): 9-14.
• Stemmer W. 1994. Rapid evolution of a protein in vitro by DNA shuffling. NatureÊ370: 389-391.
• Wilkins B. 2002. Plasmid promiscuity: meeting the challenge of DNA immigration control. Environ Microbiol. 4: 495-500.

Please send comments, suggestions, or questions to smaloy@sciences.sdsu.edu
Last modified July 5, 2004