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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
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