Gene Manipulation

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Gene Manipulation

• David R. Bell
• Room B18
• Ext 13210
• david.bell_at_nottingham.ac.uk

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Gene manipulation

• A wide variety of techniques to manipulate
  nucleic acids these techniques are
  revolutionary.
• Aim
• To understand basic techniques of molecular
  biology
• To understand the application of these techniques
  throughout biology
• i.e. the advantages of these techniques

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Limitations of protein technology

• Difficult to detect some proteins directly
• Enzyme activity can be sensitive (mg)
• Some proteins present in minute amounts
• Antibody technology very sensitive and specific
  (ng level)
• requires pure protein to raise antibodies

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Limitations (II)

• Difficult/ impossible to purify some proteins
• low amounts present, eg in development
• purification technology difficult, eg membrane
  proteins
• protease activity, therefore require fresh tissue
• fresh human tissue is rare
• many highly related proteins

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Limitations (III)

• Purification is the only source of protein
• Protein function is difficult to determine
• Difficult to study proteins in detail
• Inter-individual variation difficult to study
• Protein sequencing is difficult
• Protein studies non-definitive

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DNA - basics
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5
Base
O
CH2
Double stranded genetic material
1
4
3
2
Base
O
CH2
3
Pi
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DNA basics (II)

• 2 antiparallel complementary strands
• Adenosine base pairs with thymidine
• Cytidine with guanosine AT is weaker than CG
• DNA is coding, and unique for each species

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DNA physical properties

• The sugar-phosphate backbone is hydrophilic, but
  bases are hydrophobic, and dislike the aqueous
  environment
• ssDNA in water is stabilised by becoming double
  stranded (ds), as the bases interact with each
  other
• Annealing two ssDNA strands forming a dsDNA helix

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DNA-physical properties

• Melting a dsDNA helix coming apart
• Tm the temperature at which half of a DNA
  molecule has unwound
• Tm varies with species, and salt concentration

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Restriction enzymes
Some viruses can grow on E. coli. Virus prepared
in strain C grows inefficiently on strain
K. Once grown on strain K, virus grows
efficiently on strain K. This efficiently grows
on strain C, and is then unable to grow on
strain K again. This is known as host
restriction, and is caused by the presence of
host-specific nucleases which digest foreign DNA.
These are the restriction endonucleases. The
nuclease is accompanied by a nuclease
protection system, frequently a methylase.
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Type II R.E.s recognise a palindromic site with
rotational symmetry. Named after a bug, e.g.
EcoRI from E. coli.
5- GAATTC -3 3- CTTAAG -5
Simple cofactor require- ments- easy to do EcoRI
recognises a 6 bp site. This will occur by
chance every 46 bp, ie every 4096 bp. An enzyme
with a 4 bp site will cute every 256 bp. All
EcoRI sites are identical.
Eco RI Mg2, 37 C NaCl, pH 7
5- G -3OH 3- CTTAA -5Pi
5 Pi- AATTC -3 3OH- G -5
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Ligation of cohesive ends
All EcoRI sites give rise to exactly the
same cohesive ends.
Cohesive ends make this process
highly efficient. The process also works with
other restriction enzymes. Ligation of blunt
ends is not efficient.
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Joining DNA molecules
EcoRI site
Plasmid
Add EcoRI
Ampr
Ori
Foreign DNA
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Joining DNA (II)
Ampr
Ori
Add T4 DNA Ligase
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Joining DNA (III)
Self-ligated insert
E
Ori
E
Ampr
Insert ligated into plasmid vector now attached
to Ori and Ampr
Self-ligated plasmid
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Ligation efficiency

• Integrity of cohesive ends
• Concentration of DNA
• relative amounts of plasmid/ insert
• high concentration leads to concatenation
• Temperature
• 37 C enzyme works well, sticky ends melt
• 16 C enzyme works, sticky ends anneal

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Transformation

• The introduction of DNA into bacteria
• Incubate E. coli in CaCl2
• Yields 105-109 transformed bacteria per mg of DNA
• Electroporation- a high voltage pulse creates
  holes in E. coli
• Yields 108-1010 transformants per mg of DNA

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Selection

• Use ampicillin sensitive bacteria
• Transform bacteria with plasmid containing ori
  and ampr genes
• The only cells which grow will have the plasmid
  containing ori, ampr and the rest of the DNA in
  the plasmid.
• Clonal growth of bacteria containing one piece of
  insert DNA in a plasmid.

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Alkaline Phosphatase

• Removes 5 phosphate groups, and prevents
  ligation
• phosphatase the vector- it cannot anneal to
  itself, but can anneal to non-phosphatased DNA-
  eg the insert
• Typical ligation yields 1 pg-100ng of ligated DNA

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Alkaline Phosphatase II
Vector
Insert
OH
P
OH
P
OH
P
P
OH
Ligate
Ori
OH
P
OH
P
P
OH
P
P
OH
P
OH
OH
P
OH
P
OH
Ori
Ori
insert ligate to insert
Vector ligate to self
Insert in vector
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Alkaline Phosphatase III
Vector
Insert
OH
P
OH
OH
OH
OH
P
OH
Ori
Ligate
OH
OH
P
P
OH
P
OH
OH
OH
P
OH
P
Ori
insert ligate to insert
Insert in vector
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Cloning- the key

• The principal benefit of inserting DNA into a
  plasmid, is that it can then be put into a bug.
  The (selected) bug is grown clonally.
• Any volume of bacteria can be grown, and hence
  any amount of the one, clonal DNA molecule, or
  protein derived therefrom, can be produced......

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Summary

• Problems with protein technology
• Basic structure of DNA
• Restrictions enzymes and ligase are the tools for
  cutting and pasting DNA
• Any foreign DNA can be cut and pasted into a
  plasmid, transformed into bacteria, and then
  grown up clonally.