T7 DNA Polymerase
(E.C. 2.7.7.7)
Source:
E. coli strain containing overproducing clones of T7 Gene 5 and E. coli thioredoxin.
Description:
T7 DNA Polymerase consists of two subunits, one encoded by the bacteriophage gene 5 and the other by the E. coli trxA gene thioredoxin(1,2). This enzyme is responsible for the rapid replication of bacteriophage T7 DNA during its infection cycle. In addition to the highly processive DNA polymerase activity, it also has high levels of both single and double-stranded DNA 3'→5' exonuclease activities(3,4). The exonuclease specific activities have been reported to range from 5% to 100% of the polymerase specific activity(3,5). The exonuclease activity appears to be responsible for the high fidelity of this enzyme and prevents strand displacement synthesis(6). T7 DNA Polymerase is a valuable enzyme where high exonuclease activity is required or where absence of strand-displacement activity is
important(6). It has also been used successfully for site-directed mutagenesis(7). This enzyme is not suitable for use in DNA sequencing. It is distinct from Sequenase Version 2.0 DNA Polymerase which is a modified form of T7 DNA Polymerase with no 3'→5' exonuclease activity.
Properties:
T7 Gene 5 product (84 kDa) and E. coli thioredoxin (12 kDa).
Purity:
Greater than 95% pure as determined by SDS-PAGE. Tested for contaminating endonucleases.
Storage Buffer:
20mM potassium phosphate (pH 7.4), 1mM DTT, 0.1mM EDTA, 50% glycerol.
Assay Conditions:
The reaction mixture contains 88mM potassium phosphate (pH 7.5), 6.7mM MgCl2,
5mM 2-ME, 300μM of each dNTP, 5mM activated DNA and enzyme.
Unit Definition:
One unit is the amount of enzyme required to catalyze the incorporation of 10 nmol of total nucleotide into acid insoluble form in 30 min at 37°C under standard assay conditions.
Concentration:
10 units/μl
Tested User Friendly™ Functional Test:
Fill-in of 3' recessed ends with >50% incorporation of radiolabeled dATP into 0.1 - 0.4 μg of restricted DNA in 15 min at 30°C.
PROTOCOL FOR SECOND STRAND SYNTHESIS/MUTAGENESIS
During the synthesis reaction the mutagenic primer is extended such that the entire
template strand is copied.
10 μl annealed primer-template (0.4 pmol template/2 pmol primer)
4 μl 5X T7 DNA Polymerase Reaction Buffer
1 μl 5mM dNTP mix
1 μl 2.5 units diluted T7 DNA Polymerase
1 μl 5 units T4 DNA Ligase (PN 70005)
3 μl H2O
Incubate 60 min at 37°C. Incubate 10 min at 70°C to stop the reaction.
Functionally Tested 5X T7 DNA Polymerase Reaction Buffer (1 ml included, PN 70097):
200mM Tris-HCl (pH 7.5), 100mM MgCl2 and 250mM NaCl.
T7 DNA Polymerase Dilution Buffer (1 ml included, PN 70106):
20mM potassium phosphate buffer (pH 7.4), 1mM DTT, 0.1mM EDTA, 50% glycerol.
References:
- GRIPPO, P. AND RICHARDSON, C. C. (1971) J. Biol. Chem. 246, 6867-6873.
- MODRICH, P. AND RICHARDSON, C. C. (1975) J. Biol. Chem. 250, 5515-5522.
- ADLER, S. AND MODRICH, P. (1979) J. Biol. Chem. 254, 11605-11614.
- HORI, K., MARK, D. F. AND RICHARDSON, C. C. (1979) J. Biol. Chem. 254, 11598-11604.
- ENGLER, M. J., LECHNER, R. L. AND RICHARDSON, C.C. (1983) J.Biol.Chem. 258, 11165-11173.
- LECHNER, R. L. AND RICHARDSON, C. C. (1983) J. Biol. Chem. 258, 11185-11196.
- BEBENEK, K. et al. (1989) Nucl. Acids Res. 17, 5408.
Applications:
- Labeling 3'-termini of DNA fragments with protruding 5' ends (Fill-In Reaction).
- Labeling the 3'-termini of blunt-ended DNA fragments or the termini of DNA fragments with protruding 3'-termini (Exchange Reaction).
- Site-directed mutagenesis.
- Not suitable for DNA sequencing due to high exogenous levels of exonuclease activities.
Storage:
Shipped on dry ice. Store at -20°C.