RESTRICTION ENZYMES

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RESTRICTION ENZYMES

• GIAO NGUYEN
• ANHXUAN NGUYEN
• KATHY SZENIAWSKI

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Restriction Enzymes

• Found by Hamilton Smith and his colleagues
• Over 1500 restriction enzymes found.
• Restriction Enzymes are
• Proteins
• DNA-cutting-enzymes
• Also know as Restriction Endonucleases
• Isolated from bacteria and blue-green algae
• One of the first enzymes from E. coli.
• Named according to organisms isolated
• Example EcoRI

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Restriction Enzymes

• Cleave phosphodiester bonds of double stranded
  DNA
• Cut at specific recognition sites
• Recognize palindromic sequences of 4 to 6 base
  pair long.

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• Types of Restriction Enzymes
• Type I, II, and III
• Type I and III
• have both restriction and modification activity
• cut DNA at sites outside of their recognition
  sequences. (need ATP for energy and lack
  predictability)
• Type II RE are ideal for biotechnology
• Restriction activity but no modification
• Each cuts in a predictable manner, at a site
  within or adjacent to the recognition sequence
• Only require Mg as a cofactor, not ATP

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Two major ways RE cleaves

• blunt ends
• sticky ends

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Blunt ends
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Sticky ends
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Application

• Recombinant DNA technology
• Transformation and Selection
• Mapping and Sequencing
• Molecular Cloning

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CLEAVE WITH RESTRICTION ENZYMES, INSERT GENE OF
INTEREST INTO PLASMID
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• NOT ALL PLASMIDS TAKE UP THE GENE OF INTEREST
• NOT ALL BACTERIA TAKE UP THE PLASMIDS

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THE PLASMID IS INSERTED INTO A BACTERIAL CELL
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• HOW DO YOU INSERT THE PLASMID INTO THE BACTERIA?

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• ELECTROPORATION
• High concentration of plasmid DNA added to a
  suspension of protoplasts and the mixture is
  shocked with an electric field

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• Shotgun Cloning (1micrometer beads coated with
  DNA are shot directly into cells)

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SORT AND GROW

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BACTERIA REPLICATES, CREATING SEVERAL COPIES OF
THE GENE OF INTEREST
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WORKS CITED

• TORTORA, FUNKE CASE. 1992. Microbiology. 4th
  ed. Redwood City, CA Benjamin/Cummings.
• RAVEN, JOHNSON. 2002. Biology. 6th ed. New York.
  McGraw-Hill.
• NORFLUS, F. 2007. Biotechnology Restriction
  Enzymes. Morrow (GA) Clayton State University
• Kim,Y., Cha, J.,Chandrasegaran, S. 1996. Hybrid
  restriction enzymes Zinc finger fusions to Fok I
  cleavage domain. Biochemistry, 93, 1156-1160.
• Krane, D., Raymer, M. 2003. Fundamental Concepts
  of Bioinformatics. Pearson Education, Inc., 20.
• What are Restriction Enzymes? 2003. DNA Go to
  School. 18 January 2008. http//www.odnavaiaescola
  .com/enzymes.pdf
• Jordan, J. 2007. Biotechnology Restriction
  Enzymes. Morrow (GA) Clayton State University.

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GRAPHICS OBTAINED FROM THE FOLLOWING WEBSITES

• www.med.nyu.edu
• www.ghs.gresham.k12.or.us
• www.USERS.RCN.COM
• WWW.NCBI.NLM.NIH.GOV