Endonucleases DNA-specific

dsDNase for removal of contaminating DNA from PCR master mixes

Endonucleases DNA-specific

Endonucleases DNA-specific, dsDNase

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Double-Strand Specific dsDNase (dsDNase) is ideal for fast and effective removal of contaminating DNA from PCR master mixes.

Taq polymerases are commonly contaminated by bacterial DNA. This is a problem in PCR based bacterial typing and detection as it might cause false positive results. The unique properties of dsDNase make it suited for removal of contaminating DNA from PCR master mixes prior to addition of DNA template.

In figure 1, a PCR master mix was treated with different amounts of dsDNase before performing a qPCR to measure the contaminating bacterial DNA in the master mix. ArcticZymes dsDNase effectively removed contaminating DNA below known levels of the assay detection limits.

The dsDNase from Arctic shrimp (Pandalus borealis) is recombinantly produced in Pichia pastoris. It cleaves phosphodiester linkages in DNA to yield oligonucleotides with 5’-phosphate and 3’-hydroxyl termini.

The specific activity is estimated to be 30 times higher than that of bovine DNase I. In the presence of magnesium as only divalent cation and using oligos as a substrate, the activity towards dsDNA is 5000-fold higher than towards ssDNA.

The unique double strand-specificity allows specific degradation of dsDNA while leaving shorter ssDNA as primers and probes essentially intact. Easy inactivation by moderate heat (65°C) allows addition of DNA intended for analysis directly after removal of contaminating DNA.

Workflow – Decontamination of PCR master mixes

Recommended applications:

Key advantages with dsDNase:

Double-strand DNA specific endonuclease
High specific activity
Can be heat-inactivated by moderate heat treatment (65°C for 15 minutes)
Producing 5′-phospho-oligonucleotide products

Figures

Figure 1. The dsDNase effectively removes contaminated DNA

The dsDNase effectively removes contaminated DNA:

A PCR master mix was preincubated with various concentrations of dsDNase. After treatment, no DNA was amplified in non-template controls.

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Figure 2. HL-dsDNase effectively removes gDNA

HL-dsDNase removes at least 50 ng of gDNA in a 10 µl reaction volume at pH 7.5.

Figure 3. HL-dsDNase treatment has minimal impact on RNA integrity

RNA incubated in buffer (lanes 1-4), water (lanes 5-8) or 0.1 U/µl HL-dsDNase (lane 9-12). Samples were analysed using Bio-Rad Experion™ RNA Eukaryote Total RNA StdSens Assay.

Properties

Specificity towards double-stranded DNA

Nucleic acid specificity has been tested towards double- and single-stranded DNA and RNA oligonucleotides. The specificity of dsDNase towards the substrate has been measured using 15-mer oligonucleotides with FAM at 5′ and DarkQuencher® 3′ (Eurogentec). The fluorescence is proportional to enzyme activity. Assay conditions: 25 mM Tris pH 7.5, 5 mM MgCl₂, and 2 μM oligonucleotide.

Relative activities

Substrate Relative Activity
dsDNA                           100%
ssDNA                          <0.03%
dsRNA                          <0.01%
ssRNA                           <0.01%

Unit Definition

One unit is defined as an increase in absorbance at 260 nm of 0.001 per minute, using 50 µg/ml of high MW DNA in 100 mM Na-acetate pH 5.0 and 5 mM MgCl₂.

Specific Activity

Ca. 400 000 Kunitz Units/mg.

Activity

The HL-dsDNase is highly active in a temperature range of 20-40°C. It needs at least 2.5 mM Mg for activity and has an optimal pH at 7.5..

Storage Buffer

20 mM Tris-HCl pH 7.5, 2 mM MgCl₂, 10 mM NaCl, 0.01% (v/v) Triton X-100, 50% (v/v) glycerol.

Purity

dsDNase is purified to apparent homogeneity.

Sample Requirement

Purification of nucleic acids prior dsDNase treatment.

Storage

Minimum shelf life is 3 years at -20°C. Storage at 4°C is possible for at least 6 months. The enzyme also tolerates multiple freeze-thaw cycles.

Recommended Protocols

Ordering Info

Quantity

Product Name

SKU

Pack Size

Concentration

Price

T7 RNA Polymerase

74100-201

10 000 U

50 U/µl

$ 149.00 USD

Endonucleases DNA-specific