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Nucleic Acids Research, 1984, Vol. 12, No. 7 3155-3171
© 1984

ENZYMOLOGY

Misincorporation during DNA synthesis, analyzed by gel electrophoresis

Greg G. Hillebrand+, Alan H. McCluskey, Katherine A. Abbott, Galina G. Revich and Kenneth L. Beattie*

Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine Houston, TX 77030, USA

*To whom correspondence should be addressed

Received January 3, 1984. Revised March 12, 1984. Accepted March 12, 1984.

A method has been developed for simultaneous comparison of the propensity of a DNA polymerase to misincorporate at different points on a natural template–primer. In this method elongation of a [5'–12P]primer, annealed to a bacteriophage template strand, is carried out in the presence of only three dNTPs (highly purified by HPLC). Under these corditions the rate of primer elongation (monitored by gel electrophoresis/autoradiography) is limited by the rate of misincorporation at template positions complementary to the missing dNTP. Variations in the rate of elongation (revealed by antoradiographic banding patterns) reflect variations in the propensity for misincorporation at different positions along the template. The effect on primer elongation produced by addition of a chemically modified dNTP to ‘minus’ reactions reveals the mispairing potential of the modified nucleotide during DNA synthesis. By use of this electrophoretic assay of misincorporation we have demonstrated that the fidelity of E. coli DNA polymerase I varies greatly at different positions along a natural template, and that BrdUTP and IodUTP can be incorporated in place of dCTP during chain elongation catalyzed by this enzyme.

+Present address: Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA


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