A Virtually RNase-free DNase I
Recombinant DNase I (rDNase I)
|Digestion of DNA to undetectable levels is important in many molecular biology applications. For instance, accurate quantitation of RNA targets by RT-PCR requires the removal of contaminating genomic DNA targets. In vitro transcribed RNA is also frequently treated with DNase to remove template DNA in both research and pharmaceutical applications. Ambion now supplies a recombinant DNase I that is prepared in a host with little to no RNase activity.|
Isolating RNase-free DNase I is a Challenge
A Pure Source of DNase I
Safety of biological products is also an emerging issue. Eliminating animal tissues and animal-derived components from the production process prevents potential transmission of pathogens. Materials derived from bovine sources are of special concern, due to known transmission of bovine spongiform encephalopathy (BSE) to humans via ingestion of cow products. Regulatory agencies are increasingly restrictive in this regard, prompting the pharmaceutical industry to remove high risk raw materials from manufacturing. Ambion's rDNase I now addresses these issues by providing a recombinant replacement for pancreatic bovine DNase I.
rDNase I Performance
The recommended reaction conditions are the same as Ambion's standard DNase I. Incubate 1-2 U of rDNase I per 1 µg DNA for 30 min at 37°C in a buffer consisting of 10 mM Tris-HCl, pH 7.5, 2.5 mM MgCl2, 0.5 mM CaCl2.
Quantitative Removal of Contaminating DNA
Figure 1. Real-time PCR of Genomic DNA Samples Treated with rDNase I vs. Bovine Pancreatic DNase I. (A) Standard curve generated with 10 fold serial dilutions of human genomic DNA ranging from 100 ng to 10 pg using an ABI 7900 real-time PCR machine. The target amplified was human GAPDH. (B) GAPDH was amplified from three human genomic DNA samples (0.5 µg), either treated with DNase I (Ct=36.1), treated with rDNase I (Ct =36.9), or untreated (Ct= 23.1). The two DNase I treatments gave essentially identical results and were therefore averaged and compared to the untreated sample. The Ct of the averaged DNase I treatments vs. untreated human genomic DNA was 13.4.