Taq DSC 2.0 DNA Polymerase

• OEM by QIAGEN offers customized bulk manufacturing of this enzyme.
• Thermostable 5' -> 3' DNA polymerase
• Lacks 3ʹ→5ʹ proofreading function

Taq DSC 2.0 DNA Polymerase provides a novel, nucleic acid-based hot start, enabling high-performance PCR.

The enzyme is supplied in 20 mM Tris-HCl, 100 mM NaCl, 1.0 mM DTT, 0.1 mM EDTA, Stabilizer and 50% glycerol; pH 7.5 at 25°C.

The 10X PCR Buffer l (B7030) contains 100 mM Tris-HCl, 500 mM KCl and 15 mM MgCl₂; pH 8.3 at 25°C

OEM by QIAGEN offers customized bulk manufacturing of this enzyme.

Taq DSC 2.0 DNA Polymerase is thermostable, processive 5ʹ→3ʹ DNA polymerase. The 94 kDa protein possesses an inherent 5ʹ→3ʹ nick-translation moiety and lacks a 3ʹ→5ʹ proofreading function. The DSC formulation contains a nucleic acid based hot-start additive designed to sequester the polymerase during reaction setup and during low-temperature cycling reaction phases.

The hot-start polymerase market has been split into two major segments based on the hot-start method: antibody or chemical. Antibody methods are preferred for their rapid reactivation time, minimal cycle times and increased success rates as the weaker association of an antibody with the polymerase yields more units of active polymerase instantly. Previously, chemical methods provided advantages over antibody methods, as complete inactivation allows reaction mixtures to remain inactive for multiple days, despite their longer reactivation, which can be up to 10 minutes. This allows automation and stacking of reactions, something that — until now — has been out of reach of antibody-based methods.

Polymerase Properties

• Storage temperature: –25°C to –15°C
• Proofreading (3' ->5' exo): No
• Nick-translation (5' -> 3' exo): Yes
• Strand displacement: No
• Molecular weight: 94 kDa

Source of recombinant enzyme protein
A recombinant E. coli strain carrying the Taq DNA polymerase gene from the thermophilic organism Thermus aquaticus YT-1.

Unit definition:
One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in 30 minutes at 75°C.

Instructions
Reactions are typical µL.
On ice, prepare each of following master mixes, combine, and place in heated (94°C) thermal cycler.

2x DNA/oligonucleotide master mix:
1.0 µL 10 mM dNTPs
1.0 µL 10 µM Forward Primer
1.0 µL 10 µM Reverse Primer
1.0 µL 500 ng/µL genomic DNA
21 µL Type I Water

2x enzyme/buffer master mix:
5.0 µL 10x PCR Buffer I
0.2 µl 5 U/µL Taq DSC 2.0 DNA Polymerase
19.8 µL Type I Water

Notes
Taq DNA Polymerase is the original and most commonly used PCR enzyme. Taq excels at amplifying short (<5 kb) sequences from low-complexity template sources and produces robust yields with little or no optimization of reaction conditions.

Consider the following guidelines when designing PCR strategies using Taq DSC 2.0 DNA Polymerase.

1. DNA template: Although extensive purification of PCR templates is typically not necessary, care should be taken with crude or partially purified DNA sources as handling and chemical agents can adversely affect the PCR process. Exposure to short-wave UV light or other DNA damaging agents should be avoided, as should exposure to high ionic strength, detergents such as SDS, loading dyes and phenol. To prevent contamination from previous PCR, consider setting up reactions in a positive-pressure hood and with aerosol barrier pipet tips. In a typical 25 cycle PCR, 104 copies of target sequence will yield reproducible amplification product. This corresponds to roughly 0.1–1 ng/mL (final concentration) of plasmid DNA, and 1–10 µg/mL of genomic DNA. The use of lower DNA concentrations typically produces less non-specific product, wherase higher concentrations can allow for fewer cycles and lower mutation rates.
2. Primer design: Ideally, oligonucleotide primers are 15–30 bases in length, nearly 50% G+C, and have equal (+/– 3°C) annealing temperatures. The use of software to detect self-complementary or hairpin-prone regions is advised and is offered as a service by some synthesis providers. Note that although the 5ʹ-terminus of the primer may contain untemplated sequence, the 3ʹ end must match perfectly. Typical oligonucleotide concentration in the reaction is 0.1–0.5 µM.
3. Magnesium: Magnesium is a critical component of PCR, although its concentration can be modulated to promote various effects. Generally, 1.5–2.0 mM Mg²⁺ is targeted, but higher concentrations (up to 5 mM) may be used to stimulate the yield of reactions at the expense of fidelity. The converse is also true: lower magnesium concentrations will promote higher fidelity products with a lower overall amplification yield. Note that certain reaction components, in particular template DNA and oligonucleotides, may contribute chelating agents to the reaction which could lower the effective magnesium concentration and starve the reaction.
4. dNTPs: Generally, a final concentration of 100–200 µM dNTPs is employed, though higher concentrations may stimulate yields (particularly with longer targets) and lower may offer increases in fidelity. Taq DSC 2.0 DNA Polymerase can also incorporate and read through deoxyuridine and inosine, two analogs used in certain applications.
5. Taq DSC 2.0 Polymerase: One unit/50 µl reaction (20 U/ml) is typical, although additional enzyme may be added to stimulate yields. Taq DSC 2.0 DNA Polymerase extends a DNA template at approximately 1–2000 nucleotides/minute, so it is recommended that 30–60 seconds of extension time be provided per kb, per cycle. Appropriate extension temperatures range from 68–72°C. Because Taq DSC 2.0 DNA Polymerase exploits the natural affinity of a DNA polymerase for a duplex DNA fragment to promote its hot-start function, it does not require an extensive initial denaturation step to activate the polymerase.

Quality control analysis

Specific activity was measured using a twofold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer ([Taq-DSC 2.0]f = 0.01-0.00008µg/µl) and added to 50 µl reactions containing 10 µg calf thymus DNA, 25 mM TAPS (pH 9.3), 50 mM KCl, 2.0 mM MgCl₂, 1 mM DTT, 4 mCi/ml 3H-dTTP and 100 µM dNTPs. Reactions were incubated 10 minutes at 75°C, placed on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26).

Protein concentration is determined by OD₂₈₀ absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• Routine PCR and RT-PCR
• qPCR
• Multiplexing