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RESTRICTION ENZYMES

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Isolated from bacteria and blue-green algae. One of the first enzymes from E. coli. ... Biochemistry, 93, 1156-1160. Krane, D., Raymer, M. 2003. Fundamental ... – PowerPoint PPT presentation

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Title: RESTRICTION ENZYMES


1
RESTRICTION ENZYMES
  • GIAO NGUYEN
  • ANHXUAN NGUYEN
  • KATHY SZENIAWSKI

2
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

3
Restriction Enzymes
  • Cleave phosphodiester bonds of double stranded
    DNA
  • Cut at specific recognition sites
  • Recognize palindromic sequences of 4 to 6 base
    pair long.

4
  • 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

5
Two major ways RE cleaves
  • blunt ends
  • sticky ends

6
Blunt ends
7
Sticky ends
8
Application
  • Recombinant DNA technology
  • Transformation and Selection
  • Mapping and Sequencing
  • Molecular Cloning

9
CLEAVE WITH RESTRICTION ENZYMES, INSERT GENE OF
INTEREST INTO PLASMID
10
  • NOT ALL PLASMIDS TAKE UP THE GENE OF INTEREST
  • NOT ALL BACTERIA TAKE UP THE PLASMIDS

11
THE PLASMID IS INSERTED INTO A BACTERIAL CELL
12
  • HOW DO YOU INSERT THE PLASMID INTO THE BACTERIA?

13
  • ELECTROPORATION
  • High concentration of plasmid DNA added to a
    suspension of protoplasts and the mixture is
    shocked with an electric field

14
  • Shotgun Cloning (1micrometer beads coated with
    DNA are shot directly into cells)

15
SORT AND GROW

16
BACTERIA REPLICATES, CREATING SEVERAL COPIES OF
THE GENE OF INTEREST
17
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.

18
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
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