Colorimetric detection of DNA polymerase activity after sodium dodecyl sulfate polyacrylamide gel electrophoresis.
A nonradioactive method is developed to detect DNA polymerase activity after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis containing gapped DNA as template. The technique is based on the use of digoxigenin- or biotin-labeled deoxynucleotides during DNA synthesis, and their detection by means of an anti-digoxigenin antibody-alkaline phosphatase conjugate or by a streptavidin-alkaline phosphatase conjugate. The detection of the DNA polymerase catalytic subunit is achieved after incubation of the gels with colorimetric alkaline phosphatase substrates. The technique is able to detect nanogram amounts of Escherichia coli DNA polymerase I and picogram amounts of its Klenow fragment. The results with other DNA polymerases and E. coli extracts suggest that this colorimetric detection system could be used for the analysis of an extended range of DNA polymerase enzymes. The method presented in this report offers an alternative to the already described radioactive techniques for detection of DNA polymerase activity after SDS-polyacrylamide gel electrophoresis.
Venegas
J
J
Departamento de Bioquímica, Facultad de Medicina, Universidad de Chile, Santiago.
Solari
A
A
D003124:Colorimetry; D004259:DNA-Directed DNA Polymerase / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D012967:Sodium Dodecyl Sulfate
D012967:Sodium Dodecyl Sulfate; D004259:DNA-Directed DNA Polymerase