Methods in Molecular Biology • 16 Enzymes of Molecular Biology

52 Landgraf and Wolfes

• The enzyme is highly processive;


• Secondary structures are suppressed at the optimal temperature range
  of 70-80°C;


• dc7GTP is accepted as building block; this nucleotide analog suppresses
  secondary structures as well; and


• Taq polymerase is less expensive than T7 polymerase and its modified
  form, sequenase.
  In asymmetric PCR (8) and in single-strand sequencing in the M 13
  system (92), ddNTPs are accepted with different efficiencies com-
  pared to dNTPs. Optimal are dNTP/ddNTP ratios of 1:32 (dATP/
  ddATP), 1:16 (dCTP/ddCTP), 1:6 (dGTP/ddGTP), and 1:48 (dTTP/
  ddTTP) (8).

References

1. Chien, A., Edgar, D. B., and Trela, J. M. (1976) Deoxyribonucleic acid poly-
   merase from the extreme thermophile thermus aquaticus. J. Bacteriol. 127,
   1550-1557.


2. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A.,
   and Arnheim, N. (1985) Enzymatic amplification of [3-globin genomic se-
   quences and restriction site analysis for diagnosis of sickle cell anemia. Sci-
   ence 230, 1350-1354.


3. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T.,
   Mullis, K. B., and Erlich, H. A. (1988) Primer-directed enzymatic amplifica-
   tion of DNA with a thermostable DNA polymerase. Science 239, 487-491.


4. Koshland, D. E. (1989) The molecule of the year. Science 246, 1541.


5. Lawyer, F. C., Stoffel, S., Saiki, R. K., Myambo, K., Drummond, R., and
   Gelfand, D. H. (1989) Isolation, characterization, and expression in Escherichia
   coli of the DNA polymerase gene from Thermus aquaticus. J. Biol. Chem.
   264, 6427-6437.


6. Tindall, K. R. and Kunkel, T. A. (1988) Fidelity of DNA synthesis by the
   Thermus aquaticus DNA polymerase. Biochemistry 27, 6008-6013.


7. Gelfand, D. H. (1989) Thermus aquaticus DNA polymerase, in Current Com-
   munications in Molecular Biology: Polymerase Chain Reaction (Erlich, H.
   A., Gibbs, R., and Kazazian, H. H., eds.), Cold Spring Harbor Laboratory,
   Cold Spring Harbor, NY, pp. 11-17.


8. Innis, M. A., Myambo, K. B., Geifand, D. H., and Brow, M. A. D. (1988)
   DNA sequencing with Thermus aquaticus DNA polymerase and direct sequenc-
   ing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA
   85, 9436-9440.


9. Keohavong, P. and Thilly, W. G. (1989) Fidelity ofDNA polymerases in DNA
   amplification. Proc. Natl. Acad. Sci. USA 86, 9253-9257.


10. Dunning, A. M., Talmud, P., and Humphries, S. E. (1988) Errors in the poly-
    merase chain reaction. Nucleic Acids Res. 16, 10,393.