Cloning and genetic engineering

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• Cloning and genetic engineering
• by
• Ivo Frébort

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Cloning

• Clone a collection of molecules or cells, all
  identical to an original molecule or cell
• To "clone a gene" is to make many copies of it -
  for example, in a population of bacteria
• Gene can be an exact copy of a natural gene
• Gene can be an altered version of a natural gene
• Recombinant DNA technology makes it possible

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Plasmids

• Naturally occurring extrachromosomal DNA
• Plasmids are circular dsDNA
• Plasmids can be cleaved by restriction enzymes,
  leaving sticky ends
• Artificial plasmids can be constructed by linking
  new DNA fragments to the sticky ends of plasmid

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Cloning Vectors

• Plasmids that can be modified to carry new genes
• Plasmids useful as cloning vectors must have
• a replicator (origin of replication)
• a selectable marker (antibiotic resistance gene)
• a cloning site (site where insertion of foreign
  DNA will not disrupt replication or inactivate
  essential markers

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DNA Libraries

• Sets of cloned DNA fragments that together
  represent the genes of a particular organism
• Any particular gene may represent a tiny, tiny
  fraction of the DNA in a given cell
• Can't isolate it directly
• Trick is to find the fragment or fragments in the
  library that contain the desired gene

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Colony Hybridization

• Disk is treated with base or heated to convert
  dsDNA to ssDNA and incubated with probes
• Colonies that bind probe hold the fragment of
  interest

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Colony Hybridization
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Southern Blots
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The Polymerase Chain Reaction

• What if you don't have enough DNA for colony
  hybridization or Southern blots?
• The small sample of DNA serves as template for
  DNA polymerase
• Make complementary primers
• Add primers in more than 1000-fold excess
• Heat to make ssDNA, then cool
• Run DNA polymerase (usually Taq)
• Repeat heating, cooling, polymerase cycle

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DNA sequencingChemical Cleavage Method

• Four reactions are used
• G-specific cleavage with dimethyl sulfate,
  followed by strand scission with piperidine
• G/A cleavage depurination with mild acid,
  followed by piperidine
• C/T cleavage ring hydrolysis by hydrazine,
  followed by piperidine
• C cleavage same method (hydrazine and
  piperidine), but high salt protects T residues

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DNA sequencingChain Termination Method

• Based on DNA polymerase reaction
• Run four separate reactions
• Each reaction mixture contains dATP, dGTP, dCTP
  and dTTP, one of which is P-32-labelled
• Each reaction also contains a small amount of one
  dideoxynucleotide either ddATP, ddGTP, ddCTP or
  ddTTP

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Recombinant Proteins
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Plant transformation

• Common Strategies for Introducting Foreign Genes
  into Plant Cells
• Agrobacterium infection
• Particle bombardment
• Electroporation
• http//www.hort.purdue.edu/hort/courses/HORT250/an
  imations/Leaf20Disk20Animation/leafdisk1.html

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Regeneration of transgenic plant from
crown-gall callus
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Tobacco plants overexpressing HvCKX genes
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Transgenic Animals

• Genes can be introduced into animals by
  transfection - injection of plasmid DNA into
  recipient cells
• Plasmids can be injected into fertilized eggs in
  mice
• Expression is usually variable, because the gene
  is inserted randomly
• Growth hormone transfection produces mice that
  are very large!

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