Aptamer-Taq DNA polymerase

Product description

Description

Aptamer-Taq DNA polymerase is a modification of Combi Taq DNA polymerase (Cat. No. C205-C207) in which DNA aptamer anti-Taq is used instead of anti-Taq monoclonal antibody. When compared to antibody the aptamer has several advantages:
(1) it is not irreversibly denatured at temperature upto 100^oC,
(2) has low molecular weight, is prepared synthetically, and has well defined composition,
(3) is not contamined with components of mammalian cells,
(4) is resistant to action of proteases and allows preparation of PCR mixes at room temperature, and
(5) is less expensive.

Hot start

• This products contains DNA aptamer anti-Taq, which binds specifically to Taq DNA polymerase and blocks its enzymatic activity until the first denaturation phase of PCR. At 94^oC the antibody is irreversibly denatured and the activity of the polymerase is restored. This decreases formation of nonspecific DNA fragments during PCR.

Taq DNA polymerase

• Taq DNA polymerase is a thermostable enzyme isolated from Thermus aquaticus. The enzyme catalyzes synthesis of complementary DNA strand in the 5’->3’ direction and also possesses a 5’->3’ exonuclease activity. During amplification of DNA fragments, Taq polymerase adds at 3’ end an adenosine overhang. This can be utilized for cloning of PCR-generated DNA fragments. Advantage of the enzyme is its high processivity (amplification of 1000 base pairs takes < 1 min). Disadvantage of the enzyme is that it lacks a 3’->5’ exonuclease proofreading activity and this accounts for high error rate [about 1 error to 10⁵ - 10⁶ base pairs (bps)]. The major usage of the enzyme is in diagnostic analysis  for amplification of DNA fragments up to 5000 bps.

Technical data

Components and packaging

• Aptamer-Taq DNA polymerase is supplied at a concentration 1 U/µl. Basic packaging contains 1 tube with 500 U/500 µl (A025), 5 tubes with 500 U/500 µl (A026) or 10 tubes with 500 U/500 µl (A027).
• Each tube of Aptamer-Taq DNA polymerase is accompanied by a tube with 10x concentrated react buffer with MgCl2 (1.5 ml). If different concentration of MgCl2 is required, a tube with 10x concentrated PCR Blue Buffer without MgCl2 (1.5 ml) and a tube with 25 mM MgCl2 (0.5 ml) should be ordered (Cat. No. T059).

Storage

• At temperature -20^oC ± 5°C. Material can be repeatedly defrosted.

Composition

• Storage buffer: 20 mM Tris-HCl (pH 8.0 at 25^oC), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Nonidet P-40, 0.5% Tween 20, 50% glycerol.
• 10x reaction buffer: 750 mM Tris-HCl, pH 8.8 (at 25^oC), 200 mM (NH₄)₂SO₄, 0.1% Tween 20, 25 mM MgCl₂.

Activity

• One unit is defined as the amount of enzyme, which catalyzes incorporation of 10 nmol dNTPs within 30 min at 72^oC into trichloracetic acid precipitable material. Reaction conditions are as follow: 10 mM Tris-HCl (pH 8.8 at 25^oC), 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl₂, 200 µM dATP, dCTP, dGTP and [α-³²P]dTTP, 50 µg/ml denatured cDNA, 0.5 µM primer and 0.2 – 0.5 U of enzyme in the volume of 50 µl.

Purity and quality control

• Purity of Aptamer-Taq DNA polymerase is tested by SDS-PAGE. Enzyme migrates as a major band of 94 kDa. Material is nuclease free.
• Each batch of Aptamer-Taq DNA polymerase is tested for its ability to amplify DNA fragment from mammalian genomic DNA, isolated with DEP-25 DNA extraction kit (Cat. No. D225-D227), by PCR. The results are verified by electrophoresis in agarose gel in the presence of ethidium bromide; only DNA band of the expected size is present.