Shrimp DNase, Recombinant
Source:
Pichia pastoris strain containing overproducing clone of Pandalus borealis DNase.
Description:
Shrimp DNase is an endonuclease that cleaves phosphodiester linkages in DNA to yield di- and oligonucleotides with 5’-phosphate and 3’-hydroxyl termini. This DNase has a remarkably high specific activity towards double-stranded DNA (dsDNA). The activity towards dsDNA is 5000-fold higher than towards single-stranded DNA, and thus can be used selectively to degrade dsDNA, leaving single-stranded DNA intact. The activity of this enzyme depends on Mg2+ concentration (Fig. 1) and is stimulated by Ca2+. However, Ca2+ also stimulates the RNase activity of Shrimp DNase and should be avoided when RNA integrity is critical.
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| Fig. 1. DNase activity as a function of [Mg2+] in Tris-buffer at pH 8.0. Minimum of 2mM magnesium was required for DNase activity. Highest activity was observed at 10mM (data not shown). |
The DNase activity favors low ionic strength. Activity decreases with increasing ionic strength. This recombinant enzyme can be heat-inactivated by a moderate heat treatment without the use of EDTA (Fig. 2). Shrimp DNase is totally inactivated at 70°C after a 25-30 min incubation.
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| Fig. 2. Residual activity of Shrimp DNase. 60 units Shrimp DNase in 200 μl assay buffer was incubated at 70°C. Aliquots were taken out at indicated intervals and residual activity was measured. Shrimp DNase was totally inactivated at 70°C in 25 to 30 min. |
Properties:
Molecular Weight: 47 kDA
Optimum pH: 8.0
Km (apparent): 0.01 mg/ml
Activators: Mg2+ (Optimum: 10mM) and Ca2+ (Optimum: 1mM in the presence of Mg2+)
Inactivation: 70°C for 30 min
Ionic Strength: Low (10-20mM Tris-HCl)
Purity:
Greater than 98% pure as determined by SDS-PAGE. Tested for contaminating
ribonuclease and proteases.
Storage Buffer:
20mM Tris-HCl, pH 7.5, 2mM MgCl2, 10mM NaCl and 50% glycerol.
Assay Conditions:
The reaction mixture contains 100mM NaAC, pH 5.0, 5mM MgCl2, 50 μg/ml calf thymus DNA and Shrimp DNase. Incubation temperature is 25°C (1 ml reaction volume).
Unit Definition:
One unit increases the absorbance at 260 nm by 0.001 O.D. per min at 25°C and pH 5.0 using high molecular weight DNA as a substrate according to the method of Kunitz(1).
Concentration:
2 units/μl
References:
- KUNITZ, M. (1950) J. Gen. Physiol. 33, 349-363.
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PCR Carry Over Prevention Method is covered by US patent 6,541,204 and equivalents. A license for using the patented method is conveyed by purchase of Shrimp DNase from USB Corporation.
Applications:
- Selective degradation of dsDNA leaving ssDNA and RNA intact.
- Removal of DNA from RNA prior to RT-PCR.
- Removal of DNA template after in vitro transcription.
- Nick translation with DNA Polymerase I (PN 70010).
- Footprint determination of DNA binding protein.
Used in removal of carry-over contaminants in PCR(2).
Storage:
Shipped on dry ice. Store at -20°C.