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Taq DNA polymerase: The DNA directed DNA polymerase that has changed biotechnology

Taq DNA polymerase: The DNA directed DNA polymerase that has changed biotechnology. Sharmi W. Thor Graduate Student The University of Georgia Athens, Georgia BCMB 8010 Fall 2007.

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Taq DNA polymerase: The DNA directed DNA polymerase that has changed biotechnology

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  1. Taq DNA polymerase: The DNA directed DNA polymerase that has changed biotechnology Sharmi W. Thor Graduate Student The University of Georgia Athens, Georgia BCMB 8010 Fall 2007 Please note this presentation was created as part of an assignment for a graduate course in Advanced Biochemistry and molecular Biology BCMB8010 at the University of Georgia. Questions should be directed to Sharmi Thor: sthor@uga.edu

  2. Taq Polymerase • All organisms have DNA polymerase in order to replicate and repair DNA. • Taq polymerase was isolated from Thermus aquaticus – a thermophillic bacterium • Lawyer, F.C., Stoffel, S., Saiki, R.K., Myambo, K., Drumond, R., and Gelfand, D.H., (1989) Journal of Biological Chemistry, 264, 6427-6434 • T. aquaticus was found living in a hot spring at Yellowstone National Park • Gram –negative, filamentous, non-sporulating, non-motile, yellow pigmented bacteria • Brock, T.D., and Freeze, H., (1969) Journal of Bacteriology, 98, 289-297

  3. Structure of Taq polymerase • Molecular weight = 94 kDa • Specific activity 200,000 units/mg(20). • Single polypeptide chain • 832 amino acids Eun, H-M (1996) Enzymology Primer for Recombinant DNA Technology, Academic Press, San Diego ,California • Three domains • a 5′ to 3′ nuclease domain • an inactive 3′ to 5′ exonuclease domain (enzyme lacks proofreading ability) • 5′ to 3′ polymerase domain • Kim, Y., Eom, S.H., Wang, J., Lee, D.S., Suh, S.W., and Steitz T.A. (1995) Nature, 376, 612-616.

  4. Thumb Fingers Palm Polymerase segment Nuclease segment This diagram shows the main functional sections of Taq polymerase. The nuclease section is colored blue and the larger polymerase segment is colored red. This diagram was created using Rasmol, and Microsoft PowerPoint from data originally cited in the Protein Data Bank ID# 1taq by Kim, Y., Eom, S.H., Wang, J., Lee, D.S., Suh, S.W., and Steitz T.A. (1995) Nature, 376, 612-616.

  5. Function of Taq polymerase • Divalent cation in active site (Mg+2) • Metal ions bind to enzyme active site carboxylate residues (Asp) • One ion activates the 3′-OH on the primer for nucleophilic attack on the α-phosphate of the incoming dNTP. • The second metal ion has two jobs. It stabilizes the negative charge that builds up on the leaving oxygen and it facilitates the leaving of the β-and γ-phosphates Steitz, T.A., (1999) The Journal of Biological Chemistry, 274, 17395 -17398 • Both metal ions serve to stabilize a pentameric transition state • Steitz, T.A., (1999) The Journal of Biological Chemistry, 274, 17395 -17398

  6. Uses of Taq polymerase • Polymerase chain reaction • Incredible enzymatic thermostabiity • 9 minutes at 97.5ºC (20). • Originally, PCR used DNA pol I from E.coli Mullis, K.B. and Faloona, F. (1987) Methods in Enzymology, 155, 335-350 • PCR currently used in many areas of biological and biochemical research

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