Restriction Enzymes

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Restriction Enzymes
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Remember what we know about DNA.

• What is the monomer of DNA?
• How do bases pair?
• What kind of bond is used?

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Restriction Enzymes

• Aka Restriction Endonucleases
• What macromolecule do you think they are made of?
• Right, they are PROTEINS that cut strands of DNA
  at specific nucleotide sequences

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Restriction Enzymes

• There are many different restriction enzymes that
  each cut DNA at different nucleotide sequences
• Most will cut the DNA with a staggered cut
• Usually occurs at a palindrome

5'GAATTC 3'CTTAAG
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Sticky ends

• The staggered cuts leave the DNA with end pieces
  sticking off
• We call these sticky ends
• These exposed N-bases will want to join with
  other complimentary exposed bases

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What if???

• What do you predict could happen if two pieces of
  DNA are cut with the same restriction enzyme???
• YES! They will have the same sticky ends
• How could we use this???

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Restriction Enzymes -Kinds

• Sticky End- already discussed
• Blunt End
• These cut the DNA straight across and create
  blunt ends
• CCCGGG
• GGGCCC

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Products generated by restriction enzymes
COHESIVE END CUTTERS (staggered cuts) Enzyme
Recognition Site Ends of DNA After
Cut EcoRI 5GAATTC3 5G AATTC3 3CTTAAG
5 3CTTAA G5
PstI 5CTGCAG3 5CTGCA
G3 3GACGTC5 3G ACGTC5
BLUNT END CUTTERS (direct cuts) Enzyme
Recognition Site Ends of DNA After
Cut HaeIII 5GGCC3 5GG
CC3 3CCGG5 3CC GG5
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Restriction enzymes are named according to the
following nomenclature
In case you were curious

• Ex EcoRI
• E genus Escherichia
• co species coli
• R strain RY13
• I first enzyme isolated

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Why would anyone go through the trouble of
cutting DNA???

• One reason
• Recombinant DNA
• Break down the wordwhat do you think recombinant
  means?
• Other reasonsDNA fingerprinting, gene therapy

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• DNA that has been cut from one strand of DNA and
  then inserted into the gap of another piece of
  DNA that has been broken.
• The host DNA is often a bacterial cell such as E
  coli.

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• Bacteria are often used in biotechnology because
  they have plasmids
• A plasmid is a circular
• piece of DNA that exists
• apart from the
• chromosome and
• replicates independently of it.

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The Plasmid is then called a VECTOR

• What is a vector?
• Something that is used to transfer genes into a
  host cell
• Exs
• Bacterial
• plasmids
• Viruses

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So how do I isolate a gene of interest?

• Use a restriction enzyme!!! (duh!)

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What next???

• Once the gene is isolated, how do we join it with
  the organisms DNA?
• 1. Cut the organisms DNA with the same
  restriction enzymewhy?
• The sticky ends will naturally be attracted to
  each other
• 2. Add DNA LIGASE an enzyme that seals the
  fragments together

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What is this organism now called?

• Transgenic Organism- organisms that contain
  functional recombinant DNA (rDNA) from a
  different organism

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Whats the point?

• Recombinant DNA has been gaining importance over
  the last few years, and will become more
  important as genetic diseases become more
  prevalent and agricultural area is reduced. Below
  are some of the areas where Recombinant DNA will
  have an impact
• Better Crops (drought heat resistance)
• Recombinant Vaccines (i.e. Hepatitis B)
• Production of clotting factors
• Production of insulin
• Production of recombinant pharmaceuticals
• Plants that produce their own insecticides
• Germ line and somatic gene therapy

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RECAP

• Steps for making a transgenic organism
• Locate and isolate the gene of interest
• Cut out the gene and cut the plasmid using the
  appropriate restriction enzyme

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• 3. Insert the desired gene into the plasmid
  matching up the sticky ends

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4. Use the enzyme DNA ligase to seal up the
sticky ends
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• 5. Transfer the vector in the host organism where
  it will replicate
• 6. Host organism produces the protein coded for
  by the recombinant DNA

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Insulin Production