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Recombinant DNA (DNA Cloning)

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Recombinant DNA (DNA Cloning) SC.912.L.16.12 What is DNA cloning? When DNA is extracted from an organism, all its genes are obtained In gene (DNA) cloning a ... – PowerPoint PPT presentation

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Title: Recombinant DNA (DNA Cloning)


1
Recombinant DNA (DNA Cloning)
  • SC.912.L.16.12

2
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)

3
Whole organisms are cloned too, but differently
4
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)

5
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.

6
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
7
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.

8
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.

9
The action of a restriction enzyme, EcoRI Note
EcoRI gives a sticky end
10
Cut and Paste DNA fragments with complementary
ends (2 sticky ends)
11
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

12
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

13
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

14
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
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