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Restriction Enzymes. Remember what we know about DNA. What is the monomer of DNA? How do bases pair? What kind of bond is used?. Restriction Enzymes. Aka Restriction Endonucleases What macromolecule do you think they are made of?

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Restriction Enzymes

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Restriction Enzymes


Remember what we know about DNA.

  • What is the monomer of DNA?

  • How do bases pair?

  • What kind of bond is used?


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


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


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


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???


Restriction Enzymes -Kinds

  • Sticky End- already discussed

  • Blunt End

    • These cut the DNA straight across and create blunt ends:

    • CCCGGG

      GGGCCC


Products generated by restriction enzymes

COHESIVE END CUTTERS (staggered cuts):

EnzymeRecognition SiteEnds of DNA After Cut

EcoRI5’…GAATTC…3’5’…GAATTC…3’

3’…CTTAAG…5’3’…CTTAA G…5’

PstI5’…CTGCAG…3’5’…CTGCA G…3’

3’…GACGTC…5’3’…GACGTC…5’

BLUNT END CUTTERS (direct cuts):

EnzymeRecognition SiteEnds of DNA After Cut

HaeIII5’…GGCC…3’ 5’…GG CC…3’

3’…CCGG…5’ 3’…CC GG…5’


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


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

  • One reason…

    • Recombinant DNA

      • Break down the word…what do you think recombinant means?

    • Other reasons…DNA fingerprinting, gene therapy…


  • 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.


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.


The Plasmid is then called a VECTOR

  • What is a vector?

    • Something that is used to transfer genes into a host cell

  • Ex’s

    • Bacterial

      plasmids

    • Viruses


So how do I isolate a gene of interest?

  • Use a restriction enzyme!!! (duh!)


What next???

  • Once the gene is isolated, how do we join it with the organism’s DNA?

    1. Cut the organism’s DNA with the same restriction enzyme…why?

    • The sticky ends will naturally be attracted to each other

      2. Add DNA LIGASE: an enzyme that seals the fragments together


What is this organism now called?

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


What’s 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


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


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


4. Use the enzyme DNA ligase to seal up the sticky ends


5. Transfer the vector in the host organism where it will replicate

6. Host organism produces the protein coded for by the recombinant DNA


Insulin Production


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