Lecture Topics


Introduction

Generation of Target DNA Fragments

  • Restriction Endonuclease Digestion
    • Characteristics of restriction enzymes
    • 4-base and 6-base cutters
  • cDNA Clones
    • Comparison of prokaryotic and eukaryotic DNA
    • Stage- and tissue-specific mRNA's
    • Preparation of cDNA
  • Mechanical Shearing
  • Chemical synthesis

Insertion of Target Fragments Into Vector

  • Direct Ligation of cohesive termini
  • Blunt-end ligation
  • Linkers and adapters
  • Homopolymer tailing

Choice of Cloning Vector

  • Plasmid Vectors
    • General properties of plasmids
    • Early cloning vectors
    • pBR322
    • The pUC family of plasmids
    • Insertion of plasmids into host cells
  • Bacteriophage Vectors
    • Bacteriophage lambda
      • The lambda map
      • Identification of chimeric phages
      • size selection
      • spi phenotype
      • clear-plaque morphology
      • lac substitutions
      • biotin substitutions
      • Delivery of lambda vectors to recipient cells
    • Single-stranded bacteriophage vectors
  • Cosmids

Identification of Recombinant Clones

    • Direct Selection
    • Immunochemical Detection
    • Nucleic Acid Hybridization

Expression of Foreign DNA

  • Comparison of Transcription Initiation Signals in Prokaryotes and Eukaryotes
  • Expression Vectors
  • Lambda PL promoter
  • lacZ promoter
  • trp promoter
  • tac promoter
  • outer membrane protein promoters

Characterization of Cloned Genes

  • Restriction Mapping
  • Analysis of Protein
  • Phage infection of UV-irradiated host
  • maxicell system
  • minicell system

DNA Sequencing

  • Sanger dideoxy method
  • Cloning in Organisms other than Escherichia coli

Cloning in Bacillus subtilis

  • Transformation of Bacillus
  • Heterologous gene expression
  • Secretion vectors