DNA Polymerase I

DNA Polymerase I catalyzes the template-directed polymerization of nucleotides into duplex DNA in a 5´→3´ direction. DNA Polymerase I possesses a 3´→5´ exonuclease activity or "proofreading" function, which lowers the error rate during DNA replication, and also contains a 5´→3´ exonuclease activity, which enables the enzyme to replace nucleotides in the growing strand of DNA by nick translation. The enzyme, purified from recombinant E. coli, is capable of catalyzing de novo synthesis of synthetic homopolymers and provides a convenient method for the preparation of a variety of defined DNA substrates. DNA Pol I is provided with 10X Reaction Buffer: 500mM Tris-HCl (pH 7.2 at 25°C), 100mM MgSO₄, 1mM DTT.

Applications

• Labeling of DNA to high specific radioactivity by nick translation.
• Second-strand cDNA synthesis.

References

1. Kelly, R.B. et al. (1970) J. Biol. Chem. 245, 39–45.
2. Harwood, S.J. et al. (1970) J. Biol. Chem. 245, 5614–24.

Storage Buffer: 50mM Tris-HCl (pH 7.5 at 25°C), 1mM DTT, 0.1mM EDTA, 50% (v/v) glycerol.

Source: Recombinant E. coli strain.

QC Tests: Activity, SDS-PAGE/purity, endonuclease.

Unit Definition: One unit is defined as the amount of enzyme that incorporates 10nmol of deoxyribonucleotides into TCA-insoluble material in 30 minutes at 37°C. The reaction conditions are: 67mM potassium phosphate (pH 7.4 at 25°C), 6.7mM MgCl₂, 1mM DTT, 50μg/ml activated calf thymus DNA and 33μM dATP, dCTP, dGTP and dTTP (a mix of unlabeled and [³H]dTTP).