The metal binding ligands are also essential for the strand-displacement activity of the PRD1 DNA polymerase. Substitution of Ser or Ala for Tyr-766, at the carboxyl terminus of the long O-helix in the fingers subdomain (see Fig. Site-directed mutagenesis studies revealed that the residues important for the 3'->5' exonuclease activity, particularly metal binding ligands for the Klenow fragment, are all conserved in the PRD1 DNA polymerase as well. influence fidelity in Klenow fragment have been described. The enzyme is supplied with 10X Blue Buffer (B0110) containing 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl 2, 10 mM.
The enzyme is supplied in 20 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 50 glycerol pH 7.5 25C. Although the PRD1 DNA polymerase has a smaller 3'->5' exonuclease domain, its active sites appear to be very similar to those of the Klenow fragment. Klenow Fragment (3'5' exo-) is a mesophilic DNA polymerase that displays a moderate strand displacement activity during DNA synthesis. Note: Klenow Fragment, Exonuclease Minus, will leave a single-base 3-overhang for a significant proportion of the DNA fragments during the fill-in reaction (8). Stop the reaction by heating the mixture for 10 minutes at 75C. Incubate the reaction at room temperature for 10 minutes. In order to establish the evolutionary relationship between the family A and B DNA polymerases, we have closely compared the 3'->5' exonuclease domains between the Klenow fragment of E.coli DNA polymerase I (a family A DNA polymerase) and the bacteriophage PRD1 DNA polymerase, the smallest member of the DNA polymerase family B. unit of Klenow Fragment per microgram of DNA.
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