Klenow Fragment, exo-, is the Large Fragment of DNA Polymerase I. It exhibits 5'=>3' polymerase activity, but lacks the 3'=>5' and 5'=>3' exonuclease activities of DNA Polymerase I. The 3'=>5' exonuclease activity of the enzyme is eliminated by mutations in the 3'=>5'-exonuclease active site (1).
Source
E.coli cells with a cloned DNA fragment of the mutated polA gene.
Molecular Weight
68 kDa monomer.
Definition of Activity Unit
One unit of the enzyme catalyzes the incorporation of 10 nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30 min at 37°C, using poly(dA-dT)·poly(dA-dT) as a template·primer.
Enzyme activity is assayed in the following mixture: 67 mM potassium phosphate (pH 7.4), 6.7 mM MgCl2, 1 mM 2-mercaptoethanol, 0.033 mM dATP, 0.033 mM dTTP, 0.4 MBq/ml [3H]-dTTP and 62.5 µg/ml poly(dA-dT)·poly(dA-dT).
Storage Buffer
The enzyme is supplied in: 25 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT and 50% (v/v) glycerol.
10X Reaction Buffer
500 mM Tris-HCl (pH 8.0 at 25°C), 50 mM MgCl2, 10 mM DTT.
Quality Control
The absence of endo- and exodeoxyribonucleases confirmed by appropriate quality tests. Functionally tested in random-primed DNA labeling.
Inhibition and Inactivation
Inhibitors: metal chelators, PPi, Pi (at high concentrations) (7).
Inactivated by heating at 75°C for 10 min or by the addition of EDTA.
Note
Klenow Fragment, exo-, is not recommended for DNA blunting reactions prior to DNA ligation since it frequently adds one or more extra nucleotides to the 3'-terminus of blunt-end DNA substrates in a non-template directed fashion (8).
Klenow Fragment or Klenow Fragment, exo- or Bsm DNA Polymerase, Large Fragment
0.1 µl (1 u) 0.2 µl (1 u) 0.125 µl (1 u)
Water, nuclease-free
to 20 µl
Total volume
20 µl
Incubate at 37°C for 15 min.
Stop the reaction by heating at 75°C for 10 min.
Note
* This protocol is suitable labeling of the following Fermentas DNA markers, composed of DNA fragments with 5'-overhangs: Lambda DNA EcoRI Marker, #SM028 Lambda DNA HindIII Marker, #SM0101 Lambda DNA EcoRI/HindIII Marker, #SM0191 Lambda DNA Eco91I Marker, #SM0111 phiX174 DNA HinfI Marker, #SM0261
The modified version of this protocol can be used for non-radioactive labeling of DNA markers. Substitute a part of dTTP nucleotide with a modified nucleotide (e.g. Biotin-11-dUTP or Fluorescein-12-dUTP) at a molar ratio of 1:2.
Radioactive Random-primed DNA Labeling with Klenow Fragment, exo- or Bsm DNA Polymerase, Large Fragment
Prepare the following reaction mixture:
DNA (aqueous solution)
10 µl (100 ng)
10X reaction buffer for Klenow Fragment, exo- or 10X Bsm buffer
5 µl
6.0 A260units/ml (100 µM) Random Hexamer Primer
12.5 µl
Water, nuclease-free
to 40 µl
Total volume
40 µl
Incubate the mixture in a boiling water bath for 5-10 minutes and then chill on ice.
Add:
3 dNTP Mix, 0.33 mM each (without a labeled dNTP)
3 µl (0.02 mM final concentration)
[alpha-32P]-dNTP, ~110 TBq/mmol (3000 Ci/mmol)
1.85 MBq (50 µCi)
Klenow Fragment, exo- or Bsm DNA Polymerase, Large Fragment
1 µl (5 u) 1 µl (8 u)
Water, nuclease-free
to 50 µl
Total volume
50 µl
Incubate the reaction mixture for 10 minutes at 37°C.
Add 4 µl 0.25 mM dNTP mix and incubate at 37°C for 5 minutes.
Add 1 µl 0.5 M EDTA, pH 8.0 to stop the reaction.
Remove 1 µl of the reaction mixture and determine the percentage of label incorporated.
Purify by using Sephadex G-50 or Bio-Gel P-60.
Non-radioactive Random-primed DNA Labeling with Klenow Fragment, exo-
Prepare the following reaction mixture:
DNA template
10 µl (100 ng – 1 µg)
10X reaction buffer for Klenow Fragment, exo-
5 µl
6.0 A260units/ml (100 µM) Random Hexamer Primer
12.5 µl
Water, nuclease-free
to 39 µl
Total volume
39 µl
Incubate the mixture in a boiling water bath for 5-10 minutes and then chill on ice.
Add:
3 dNTP Mix, 1 mM each (without the dTTP)
5 µl (0.1 mM final concentration)
dTTP
3.25 µl (0.065 mM final conc.)
Biotin-11-dUTP*, 1 mM
1.75 µl
Klenow Fragment, exo-
1 µl (5 u)
Total volume
50 µl
* Fluorescein-12-dUTP, DIG-dUTP or Aminoallyl-dUTP can also be used with the same protocol.
Incubate the reaction mixture at 37°C for 1 hour.
Add 1 µl 0.5 M EDTA, pH 8.0 to stop the reaction.
Remove 1 µl of the reaction mixture and determine the percentage of label incorporated.
Optionally, purify by using Sephadex G-50 or Bio-Gel P-60.
References
Derbyshire, V., et al., Genetic and crystallographic studies of the 3’,5’-exonucleolytic site of DNA polymerase I, Science, 240, 199-201, 1988.
Feinberg, A.P., Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal. Biochem., 132, 6-13, 1983.
Feinberg, A.P., Vogelstein, B., Addendum to: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal. Biochem., 137, 266-267, 1984.
Yu, H., et al., Cyanine dye dUTP analogs for enzymatic labeling of DNA probes, Nucleic Acids Res., 22, 3226-3232, 1994.
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- Thermal inactivation at 75°C in 10 min
- Recombinant enzyme
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