Recombinant DNA

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RE Digestion
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Cloning Vectors

• Specifically modified chromosomes that are used
  to propagate foreign DNA in bacteria (E. coli) or
  yeast (S. cerivisiae).
• Three essential features for a cloning vector
• 1) Origin of replication
• 2) Dominant selectable marker
• 3) Single restriction sites
• Types
• Plasmid circular chromosomes in E. coli
• Bacteriophage E. coli virus
• Cosmid modified plasmids
• YAC yeast artificial chromosomes

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Plasmids

• Small circular DNA molecules
• Use clones of small DNA fragments (100-5,000 bp)
  maintained in E. coli cells.
• Design active origin of replication allows for
  multiple rounds of replication during each cell
  division, so many copies of the plasmid are
  present in every cell. Genes conferring
  resistance to antibiotics (ampicillin, kanamycin)
  used as selectable markers. Phenotypic markers
  used to indicate presence of foreign DNA.
  Plasmids introduced into E. coli by
  transformation, and easily purified from culture.

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Recombinant DNA
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Bacteriophage

• Modified bacterial viruses, l phage
• Use constructing and maintaining genomic or cDNA
  libraries with 5-15 kb inserts
• Design many genes normally used by the phage are
  removed and replaced with foreign DNA. Propagated
  through a bacterial (E. coli) host--infect cell,
  replicate, and lyse cell releasing 1,000s of
  phage progeny. Easy to manipulate and screen to
  identify clone of interest.

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Vectors for large inserts

• Cosmids hybrid between plasmid and phage
• Use genomic libraries with 35-45 kb inserts
• Design entry into cell by phage infection,
  followed by maintenance as a plasmid

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Vectors for large inserts

• BAC bacterial artifical chromosome
• Use organization and maintenance of a large
  genome in clones containing 150-kb inserts.
• Design chromosome containing foreign DNA with E.
  coli ori and selectable marker.
• YAC yeast artificial chromosome
• Use organization and maintenance of a large
  genome in clones containing 200-500 kb inserts.
• Design chromosome containing foreign DNA with
  yeast centromere, telomeres, ARS, and selectable
  marker.

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Subcloning
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DNA Library

• Libraries are constructed by inserting a variety
  of sequences into a cloning vector, and
  simultaneously maintaining all of these clones
  each clone representing an individual insert.
• Genomic Library randomly cut the genome into
  appropriate size for vector, mix and ligate with
  vector, replicate in host. Each clone (a vector
  with an insert) represents a unique region of the
  genome, but each region of the genome is
  represented equally in overlapping clones.
• cDNA Library isolate mRNA from a cell or tissue
  type, convert into DNA, and ligate with vector.
  Each clone represents a DNA copy of an mRNA, and
  each mRNA is represented in the library based on
  its level of transcription.

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Genomic Library
Genomic DNA
Randomly cut
Ligate
Transform
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