Description:
Sequenase™ Version 2.0 DNA Polymerase is a genetically engineered form of T7 DNA polymerase. Unlike the wild-type enzyme it has virtually no 3'→5' exonuclease activity. Sequenase Version 2.0 is highly processive, incorporates nucleotide analogs (dlTP, thio-dNTPs, dideoxy-NTPs, etc.), is not impeded by secondary structures, and can carry out strand displacement synthesis. It is an excellent enzyme for dideoxy-sequencing, and is useful in other applications, especially where the absence of associated exonuclease activity is desirable.

Sequenase Version 2.0 has two subunits, one is the E. coli protein thioredoxin (MW 12,000) and the other is a genetically engineered version of bacteriophage T7 gene 5 protein (MW 76,000). The genetic changes in this subunit (a deletion of 28 amino acids accomplished by in vitro mutagenesis) eliminate all measurable exonuclease activity without changing the DNA polymerase activity.

Properties:
Molecular Weight: Consists of two subunits, modified T7 gene 5 protein (76 kDa) and E. coli
Thioredoxin (12 kDa)
Optimum pH: 7.5
Optimum Temperature: 37°C
Requirements for Divalent Cation: Mg2⁺, Mn2⁺

Purity:
Greater than 95% pure as determined by SDS-PAGE.Tested for contaminating double- and single-stranded endonucleases and exonucleases.

Storage Buffer:
20mM potassium phosphate (pH 7.4), 1mM DTT, 0.1mM EDTA, 50% glycerol.

Assay Conditions:
The reaction mixture (100 μL) contains 40mM Tris-HCl (pH 7.5), 10mM MgCl₂, 5mM DTT, 0.3mM dNTPs, and 5 μg M13mp18 pre-annealed to 5 pmol M13 universal primer. The enzyme is added to the pre-warmed (37 °C) reaction mixture; incubation is at 37 °C for 1 min.

Unit Definition:
One unit of enzyme catalyzes the incorporation of 1 nmol of nucleotide into acid insoluble form in 30 sec at 37 °C.

Concentration:
13 units/μL

Functional Test:
DNA sequencing with the Sequenase Version 2.0 DNA Sequencing Kit (PN 70770).

Functionally Tested 5X Sequenase Reaction Buffer (1 ml included, PN 70702):
200mM Tris-HCl (pH 7.5), 100mM MgCl₂, 250mM NaCl

Sequenase Dilution Buffer (1 ml included, PN 70765):
10mM Tris-HCl (pH 7.5), 5mM DTT, 0.1mM EDTA.

References:
Tabor, S. and Richardson, C. C. (1989) J. Biol. Chem. 264, 6447-6458.
Wang, D., Coscoy, L., Zylberberg, M., Avila, P. C., Boushey, H. A., Ganem, D. and DeRisi, J. L. (2002) Proc Natl. Acad. Sci. USA, 99, 15687-15692.
Paris, M. (1992) Comments 18, (No. 3), United States Biochemical Corporation, Cleveland, Ohio.
Tabor, S. and Richardson, C. C. (1987) Proc.Natl. Acad Sci. USA 84, 4767-4771.
Tabor, S. and Richardson, C. C. (1989) Proc.Natl. Acad. Sci. USA 86, 4076-4080.