Shrimp nuclease (recombinant)

Shrimp nuclease is an enzyme with a particularly high preference for double-stranded DNA, and can be used to selectively degrade dsDNA, leaving ssDNA and RNA intact. The enzyme is also inactivated after 30 minutes at 65 °C.  Shrimp nuclease is produced in recombinant form in Pichia pastoris.

Use for removal of carry-over contaminants in PCR [1]
Since previous PCR products will be double-stranded, these can be selectively degraded using Shrimp nuclease. The enzyme can be added directly to PCR reaction mixtures before the addition of template DNA. A heat inactivation step will remove the nuclease before the addition of template.

Use for removal of genomic DNA in RT-PCR
The substrate specificity of Shrimp nuclease makes the enzyme ideal for removing contaminating genomic DNA  from RT-PCR reaction mixtures. Our enzyme has a low activity towards cDNA in RNA/DNA hybrids, and therefore the enzyme can be added directly to the complete RT mix. Contaminating DNA is removed during the reverse transcriptase step, and both the nuclease and the reverse transcriptase is inactivated by heating after the RT-step. The method is useful both for two-step and one-step RT-PCR applications.

Use for contamination control in RT-PCR [1]
Shrimp nuclease is well suited to remove contaminating carry-over PCR-products in RT-PCR. The enzyme will leave both the RNA template and the primers intact. Since the activity towards cDNA in DNA/RNA hybrid is low, the treatment will have a minimal effect on Ct values. The result is an effective decontamination protocol in a closed-tube format. An additional advantage is that proof-reading polymerases may be used, since the method does not require unnatural nucleotides. Also, no third-party licenses are required.

Use for normalisation of cDNA libraries
The preference for dsDNA can be utilized for normalization of cDNA libraries. Since abundant cDNA species will re-anneal faster after denaturation, Shrimp nuclease will selectively degrade the high-copy cDNAs. A subsequent amplification of the library will result in a normalized library.

Availability
Recombinant Shrimp nuclease is available directly from Biotec Pharmacon, from USB Corp (Affymetrix, worldwide) or Thermo Scientific (UK). 

Specifications
Unit definition: With high-molecular weight DNA as substrate, 1 Unit will cause an absorbance increase of 0.001 per minute at 260 nm. Reaction conditions: 0.05 mg/ml DNA in 100 mM Na-acetate pH 5.0, 5 mM MgCl₂, 25 °C.

Specific activity: >400,000 Units/mg

Purity: Purified to apparent homogeneity by SDS-PAGE. RNase or protease activities not detected.

Concentration: Minimum 1,000 Units/ml.

Properties
Stability: Stable at -20 °C in storage buffer. Completely inactivated after 30 min at 65°C.

Substrate specificity: Activity towards ssDNA is less than 0.03 % of dsDNase activity (depending on Mg²⁺ concentration). Intrinsic RNase activity at 5 mM MgCl₂ is comparable to commercial RNase-free DNase I preparations.

Cofactor requirements: Shrimp nuclease is dependent on Mg2+ ions for activity. Maximum activity is achieved at 10 mM Mg2+, minimum concentration required for activity is 1.5 mM.

[1] Method for removal of contaminating PCR products from PCR or RT-PCR is subject to patent rights according to US patent 6,541,204 and equivalents. A license for using the patented method is conveyed by purchase of Shrimp nuclease from Biotec Pharmacon or it's distributors.