DNase Testing

DNase, or Deoxyribonuclease, is an enzyme that catalyzes the hydrolytic cleavage of the phosphodiester backbone of DNA. It can digest single- and double-stranded DNA, chromatin, and DNA-RNA hybrids. DNases play a vital role in various cellular functions and are essential for research and biotechnological applications. Since these enzymes are ubiquitous, they can cause contamination if exposed to certain scientific processes.

History of DNase Testing

DNase activity assays have a long history. One traditional method is Agarose Gel-Based Electrophoresis, widely used for decades [1] (see figure 1). This technique involves resolving DNA fragments after digestion with DNases on an agarose gel and visualizing them with ethidium bromide staining. Other methods include colorimetric [2] and ELISA-based assays [3].


Image 1: Previously accepted DNase activity assay. In this image shown, DNA samples were incubated at 37C for 1 hour, and analzyed on 1.0% agarose gel using TAE buffer. Using reference lane 1 and 7, and DNase-free lanes 2 and 5, the DNase-affected DNA in lanes 3,4 and 6 can be examined. Agarose-Gel Electrophoresis was widely used for decades, prior to technological advancements in DNase testing.

Importance of DNase Assay for Buffers, Reagents, and Media

DNases are enzymes that can degrade DNA. They are ubiquitous in nature and are highly utilized. Their affinity for DNA is incredibly high, and often leads to the cleavage of target DNA in these environments. The presence of DNase in buffers or solutions can compromise molecular biology experiments specifically testing for DNA or targeting DNA strands, which results in skewed data that affects further research and discovery. Therefore, it is important to prepare all buffers, reagents, and media relevant to these experiments under conditions that prevent DNase contamination and test for DNase activity.


Figure 2: The mechanism of DNase in DNA cleavage. In this machanism, DNase catalyzes the hydrolysis of phosphodiester bonds in DNA molecules, resulting in the cleavage of the DNA strands. This cleavage process occurs by the enzyme binding to the DNA at specific recognition sites, typically rich in adenine (A) and thymine (T) base pairs, and subsequently breaking the phosphodiester bonds between adjacent nucleotides. DNase plays a vital role in various cellular processes, including DNA repair, gene regulation, and DNA degradation.

What Test is Used at Boston BioProducts?

Boston BioProducts uses fluorescence-labeled oligonucleotide probes that fluoresce after nuclease digestion. The increase in fluorescence intensity is directly proportional to the amount and activity of the DNase present in each sample.


Image 1: DNase Testing at Boston BioProducts. This DNase testing procedure highlights DNase detection with bright-green fluorescence.

Considerations and Limitations of DNase Testing

pH of Buffer Solutions DNase, and DNase testing is optimal at neutral pH (6-8). Understanding your buffer sample is important, especially in this higher-risk range.
High Salt Concentration High salt solutions inhibit results of DNase testing as DNase activity is halted in high salt concentration. Dilutions of these solutions is necessary prior to performing the DNase Assay.
Presence of Divalent Cations DNase activity is inhibited by divalent cations such as Zn2+ Hg2+ Pb2+, Cd2+, As2+, and Cu2+. Whereas the divalent cations such as Mg2+, Mn2+ and Ca2+ are known to activate the DNases activity.
Presence of Chelators Chelators like EGTA and EDTA inhibit DNase activity.
Reducing Agents Reducing agents like β-mercaptoethanol, dithiothreitol (DTT), dithioerythritol (DTE), and glutathione (GSH) inhibit the DNase activity as the reduced enzyme is inactive.
Detergent SDS inhibits DNase activity.
Chaotropic Agents Chaotropic agents such as guanidine thiocyanate, guanidine hydrochloride and urea inhibit the DNase activity.
Alkylating Agents Alkylating agents such as iodoacetamide (IAA) and iodoacetate inhibit DNase activity.
Gel Loading Buffers or Colored Solutions Colored solutions interfere with DNase assays, especially if they contain SDS, EDTA, or β-mercaptoethanol.

In addition to DNase Testing, Boston BioProducts provides a comprehensive set of QC tests for custom reagents. Learn more about custom reagent development services.