Recombinant DNA (DNA Cloning)

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Recombinant DNA (DNA Cloning)

• SC.912.L.16.12

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What is DNA cloning?

• When DNA is extracted from an organism, all its
  genes are obtained
• In gene (DNA) cloning a particular gene is copied
  (cloned)

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Whole organisms are cloned too, but differently
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DNA cloning and Recombinant DNA

• Major tool-restriction enzymes
• discovered in late 1960s
• cuts DNA at restriction site
• highly specific
• manufactured by bacteria
• Cloning vector - carrier for moving DNA into a
  cell such as a bacterial virus or plasmid into
  which foreign DNA can be inserted
• Recombinant DNA joining together of two
  fragments of DNA that are not normally joined
  together (e.g. joining together of eukaryotic DNA
  and prokaryotic DNA - usually in a cloning vector)

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Why Clone DNA?

• A particular gene can be isolated and its
  nucleotide sequence determined
• Control sequences of DNA can be identified
  analyzed
• Protein/enzyme/RNA function can be investigated
• Mutations can be identified, e.g. gene defects
  related to specific diseases
• Organisms can be engineered for specific
  purposes, e.g. insulin production, insect
  resistance, etc.

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How is DNA cloned?
Blood sample

• DNA is extracted- here from blood
• Restriction enzymes, e.g. EcoRI, HindIII, etc.,
  cut the DNA into small pieces
• Different DNA pieces cut with the same enzyme can
  join, or recombine.

DNA
Restriction enzymes
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Restriction Enzymes

• Bacteria have learned to "restrict" the
  possibility of attack from foreign DNA by means
  of "restriction enzymes.
• Cut up foreign DNA that invades the cell.
• Type II and III restriction enzymes cleave DNA
  chains at selected sites.
• Enzymes may recognize 4, 6 or more bases in
  selecting sites for cleavage.

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Basics of type II Restriction Enzymes

• No ATP requirement.
• Recognition sites in double stranded DNA have a
  2-fold axis of symmetry a palindrome.
• Cleavage can leave staggered or "sticky" ends or
  can produce "blunt ends.

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The action of a restriction enzyme, EcoRI Note
EcoRI gives a sticky end
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Cut and Paste DNA fragments with complementary
ends (2 sticky ends)
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DNA Cloning, II

• Bacterial plasmids (small circular DNA additional
  to a bacterias regular DNA) are cut with the
  same restriction enzyme
• A chunk of DNA can thus be inserted into the
  plasmid DNA to form a recombinant

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DNA cloning, III

• The recombinant plasmids are then mixed with
  bacteria which have been treated to make them
  competent, or capable of taking in the plasmids
• This insertion is called transformation

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DNA Cloning IV

• The plasmids have naturally occurring genes for
  antibiotic resistance
• Bacteria containing plasmids with these genes
  will grow on a medium containing the antibiotic-
  the others die, so only transformed bacteria
  survive

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PCRpolymerase chain reaction

• DNA doubled in each cycle
• starting with 1 molecule of DNA
• 1 million copies after 20 cycles
• Key to procedure is thermostable DNA polymerase
  (Taq polymerase)
• Ability to amplify minute amounts of DNA valuable
  in many disciplines
• basic and applied research
• criminal forensic science
• ecology
• analysis of ancient DNA