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
DNase I has historically been prepared from bovine pancreas, one of the richest sources of RNase activity (~1 mg RNase A per gram of tissue). Therefore, it is often hard to obtain DNase I sufficiently free of RNase that it will not compromise RNA analysis experiments. Providing a high quality, RNase-free DNase I is very important to Ambion. This is borne out in a recent Biotechniques article (Matthews et al, 2002) that compared several different manufacturer's DNase I products (see sidebar at right).
A Pure Source of DNase I
Ambion employs stringent purification protocols and qualifies our bovine-derived DNase I at concentrations 5X or greater than that recommended for use. Now Ambion goes a step further by providing an alternative to bovine DNase I, recombinant DNase I (rDNase I), that further eliminates residual RNase and other copurified contaminants. rDNase is a highly purified product derived from cloned bovine DNase I. The rDNase I is prepared in a host that has RNase levels that are 1 x 107 fold lower than bovine pancreas.
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
Ambion's rDNase I meets or exceeds the performance of native bovine DNase I. It digests DNA to oligonucleotide-sized products or shorter. Yet its reaction kinetics are identical to that of native DNase I. Thus, rDNase I can be directly substituted for native DNase I with equivalent units.
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
To demonstrate the efficiency of rDNase I in removing residual DNA, we measured genomic DNA contamination by real-time PCR before and after DNase I treatment. A total of 0.5 µg of human genomic DNA was incubated in 50 µl, and digested with 2 U of DNase I or rDNase I in a volume of 50 µl for 30 min at 37°C. One-tenth of this reaction volume (equivalent to 50 ng genomic DNA) was added to a 25 µl real-time PCR reaction. The efficiency of DNA degradation was monitored by comparing cycle thresholds (Ct
), with and without DNase digestion. As shown in Figure 1B, both DNase I and rDNase I shifted the real-time PCR signal by an average of 13.4 Ct
's, or about 11,000-fold, to less than a single copy genome equivalent (~2 pg genomic 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.
Lack of RNase Activity
Since rDNase I is derived from a substantially "cleaner" source than pancreatic DNase I, the enzyme can be more readily purified to a RNase-free state. Although the recommended usage conditions for DNase I rarely require more than 2U per reaction, rDNase I passes Ambion's stringent RNase contamination quality control assay using 10 times as much enzyme for a 32-fold longer incubation! This result means that rDNase I is qualified RNase-free at 320 times typical usage conditions, creating an unsurpassed margin of safety for every application.