Klenow Fragment exo- (3’→5′ exo-) is a product of E. coli DNA Polymerase I which lacks the N-terminal 324 amino acids. This enzyme lacks the 5’→ 3′ exonuclease activity of intact DNA Polymerase I, and has mutations (D355A, E357A) which abolish the 3´→ 5´ exonuclease activity but does exhibit the 5’→ 3′ DNA polymerase activity (1). These characteristics make the enzyme useful for several molecular biology applications such as:
- Recombinase polymerase amplification (RPA) assay
- Di-deoxy sequencing (2)
- Second strand cDNA synthesis
- Second strand synthesis in mutagenesis (3)
- Single-stranded DNA probes generation
The physical purity of this enzyme is ≥99% as assessed by SDS-PAGE with Coomassie® blue staining (see figure below).
Klenow Fragment exo-
10x Klenow exo- reaction buffer
50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM β-mercaptoethanol, 1 mM DTT and 50% (v/v) glycerol.
One unit is defined as the amount of enzyme that will incorpo-rate 10 nmol of dNTP into acid-insoluble material in 30 minutes at 37°C under standard assay conditions.
10x Reaction Buffer
500 mM Tris-HCl, 100 mM MgCl2, 50 mM dithiothreitol (DTT), pH 7.5 @ 25°C.
E. coli cells carrying E. coli polA gene without its N-terminal exonuclease domain and carry D355A/E357A mutations.
Inactivated by heating at 75°C for 20 min.
Quality Control Assays
DNA Polymerase I, Klenow Fragment exo- is free from detect-able endonuclease and RNase activities.
1. Derbyshire, V. et al. (1988). Science. 240, 199-201.
2. Sanger, F. et al. (1977). Proc. Natl. Acad. Sci. USA. 74, 5463-5467.
3. Gubler, U. (1987). In S.L. Berger & A.R. Rimmel(Ed.), Methods in Enzymology. 152, 330-335. San Die-go: Academic Press.