Description:
Sequenase™ version 2.0 DNA polymerase is a genetically engineered form of T7 DNA polymerase. Unlike the wild-type enzyme, it has almost no 3'→5' exonuclease activity. Sequenase version 2.0 is highly processive, contains nucleoside analogs (dlTP, thio-dNTPs, dideoxy-NTPs, etc.), is not hindered by secondary structure, and allows for strand displacement synthesis. This enzyme works well for dideoxy sequencing and is also useful in other applications, especially where the relevant exonuclease activity is not present.

Sequenase version 2.0 has two subunits, one is the E. coli thioredoxin (MW 12,000) and the other is the genetic engineering of the bacteriophage T7 gene 5 protein (MW 76,000) Modified version. Genetic alteration of this subunit (deletion of 28 amino acids by in vitro mutation) eliminated all measurable exonuclease activity but did not alter DNA polymerase activity.

Characteristics:
Molecular weight: consists of two subunits, modified T7 gene 5 protein (76 kDa) and E. coli
Thioredoxin (12 kDa)
Excellent pH: 7.5
Excellent temperature: 37°C
Divalent cation requirements: Mg2⁺, Mn2⁺

Purity:
Purity measured by SDS-PAGE is greater than 95%. Tests were performed for contaminating double-stranded and single-stranded endonucleases and exonucleases.

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

Detection conditions:
Reaction mixture (100 µL) contains 40 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 5 mM DTT, 0.3 mM dNTPs, and 5 µg M13mp18 (preannealed to 5 pmol M13 universal primer). Add enzyme to prewarmed (37°C) reaction mixture; incubate at 37°C for 1 minute.

Unit definition:
One unit of enzyme can catalyze the incorporation of 1 nmol of nucleotide into insoluble acid in 30 seconds at 37°C.

Concentration:
13 units/µL

Functional Assay:
Use Sequenase Version 2.0 DNA Sequencing Kit (PN 70770) Performing DNA Sequencing

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

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

References:
Tabor, S. and Richardson, CC (1989) J. Biol.Chem.264, 6447-6458.
Wang, D., Coscoy, L., Zylberberg, M., Avila, PC, Boushey, HA, Ganem, D. and DeRisi, JL (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, CC (1987) Proc.Natl.Acad Sci.USA84, 4767-4771.
Tabor, S. and Richardson, CC (1989) Proc.Natl.Acad.Sci.USA86, 4076-4080.