Deoxyribonuclease I in Advanced Molecular Biology Applications

Deoxyribonuclease I (DNase I) is a vital enzyme that acts as nature’s DNA degradation system. Its primary role is to break down the DNA from dying cells during processes like apoptosis (programmed cell death) and necrosis, which prevents the accumulation of cellular debris. This cleanup function is crucial for preventing the immune system from mistakenly attacking the body’s own DNA. Additionally, in some animals, DNase I is secreted by the pancreas to aid in the digestion of nucleic acids from food sources.

Deoxyribonuclease I (DNase I) is a versatile endonuclease that non-specifically cleaves DNA and serves as a critical enzymatic tool in molecular biology. Its utility is indispensable, spanning a diverse array of laboratory procedures where it ensures experimental accuracy and validity. This article elucidates the significant contributions of DNase I to several key research workflows, underscoring its importance for investigators in the field.

Applications of DNase I enzymes

Common applications include degrading contaminating DNA from RNA preparations, identifying protein-binding sites on DNA through footprinting, and preventing cell clumping in culture.

Genomic DNA decontamination: ensuring RNA integrity

Accurate analysis of RNA requires the exclusion of contaminating genomic DNA (gDNA). The co-purification of gDNA during RNA extraction is a common technical challenge that can generate false signals or misrepresent transcript abundance in sensitive downstream applications such as reverse transcription-polymerase chain reaction (RT-PCR), quantitative PCR (RT-qPCR) [Fig. 1], and RNA sequencing (RNA-Seq). This is particularly relevant as RT-PCR can amplify even a single molecule from a complex mixture. RNA isolated from certain tissues, such as spleen, kidney, or thymus, and from transfected cells, often contain higher levels of DNA contamination.

DNase I is the standard enzymatic agent used to degrade this contaminating DNA. The treatment can be performed during RNA isolation (e.g., on-column digestion) or after elution, where the enzyme hydrolyzes gDNA into smaller oligonucleotides. This selective removal of DNA preserves the integrity of the RNA, which is crucial for generating high-fidelity gene expression data and enhancing the reliability of transcriptomic studies.

Learn about methods to check RNA integrity >

Chromatin accessibility profiling: elucidating regulatory architecture

Chromatin accessibility profiling is a technique used to identify areas where chromatin is less condensed and regions where gene expression can start. The areas where chromatin is less condensed can be reached by DNase I and is called DHSs (DNase Hypersensitivity site).

Investigating gene regulatory mechanisms often involves assessing chromatin structure. DNase I is a pivotal reagent in methods designed to map accessible chromatin regions, known as DNase I Hypersensitive Sites (DHSs), which are frequently associated with active regulatory elements like promoters and enhancers. The principle behind this application is that euchromatic regions, which have less condensed chromatin structure, are more susceptible to DNase I cleavage.

Techniques such as DNase-Seq utilize controlled DNase I digestion of isolated nuclei. The enzyme preferentially cuts DNA within these accessible areas, and the resulting DNA fragments are then sequenced to facilitate the genome-wide identification of DHSs. These maps provide comprehensive insights into the active regulatory landscape of a cell, advancing our understanding of gene regulatory networks.

DNase footprinting: delineating protein-DNA interactions

DNase I footprinting is a well-established, high-resolution technique used to identify the precise DNA sequences bound by specific proteins [Fig. 2], such as transcription factors. When a DNA-binding protein is complexed with its target sequence, it sterically blocks DNase I from accessing and cleaving the DNA backbone in that region.

In this method, a DNA fragment of interest is end-labeled and incubated with the protein being studied. A limited digestion with DNase I is then performed. When the resulting DNA fragments are separated by electrophoresis, the protein-bound segment appears as a “footprint”—a region with few or no cleavage products compared to a control reaction lacking the protein. This allows for the precise mapping of protein-binding sites, often down to the nucleotide level.

In-vitro transcription clean-up: production of high-purity transcripts

The synthesis of RNA in vitro (IVT) produces RNA from a DNA template [Fig. 3], and it is essential to subsequently remove this template. The persistence of the DNA template can interfere with downstream applications, including its use in cell-free translation systems, microinjection studies, or as therapeutic agents like mRNA vaccines. Following the IVT reaction, DNase I is used to specifically hydrolyze the DNA template while leaving the RNA product intact. This enzymatic treatment ensures the generation of pure, DNA-free RNA, which is critical for the integrity and safety of subsequent experiments and therapeutic products.

Discover DNase I enzyme for RNA sample preparation >

Optimization of cellular assays and sample processing

DNase I is also used to mitigate technical challenges in cell culture and sample preparation, particularly for single-cell analyses. During tissue dissociation or cell handling, DNA released from lysed cells increases sample viscosity and promotes cell clumping. This aggregation can adversely affect cell viability, cause inaccuracies in cell counting, and compromise single-cell genomic data. Including DNase I in dissociation buffers or cell suspension media breaks down this extracellular DNA, promoting the creation of homogenous single-cell suspensions and thereby improving the quality and reliability of data from various cell-based assays.

Learn more about DNase treatment for RT-PCR >

DNase I enzyme characteristics and reaction conditions

Historically, DNase I has been purified from bovine pancreas, a rich source of RNases like RNase A. This makes it challenging to obtain DNase I that is sufficiently free of RNase activity, which could compromise RNA analysis experiments.

To address this, a recombinant DNase I (rDNase I) is now available, prepared in a host with little to no RNase activity. This rDNase I is a highly purified product derived from cloned bovine DNase I and eliminates residual RNase and other contaminants. This recombinant alternative also enhances safety by eliminating animal-derived components, preventing the potential transmission of pathogens like bovine spongiform encephalopathy (BSE). The performance of rDNase I meets or exceeds that of native bovine DNase I and can be directly substituted with equivalent units.

Check out recombinant RNase-free DNase I enzyme >

Cleavage specificity

While considered a non-specific endonuclease, DNase I does exhibit some sequence preference, favoring cleavage at purine-pyrimidine sequences and being sensitive to the structure of the minor groove. It digests double-stranded DNA (dsDNA) into dinucleotides, trinucleotides, and other oligonucleotides. Its activity is much lower on other substrates; for instance, its specific activity on single-stranded DNA (ssDNA) is about 500 times less than on dsDNA, and its activity on the DNA strand in an RNA-DNA hybrid is less than 2% of its activity on dsDNA.

Optimal reaction conditions for DNase I

The activity of DNase I is highly dependent on the reaction buffer composition.

• Cation Requirement: DNase I requires both magnesium (Mg2+) and calcium (Ca2+) ions for optimal activity. It is not active in buffers containing only Mg2+ without any Ca2+. Even micromolar levels of contaminating Ca2+ can act as a potent activator. Consequently, the chelating agent EDTA can inhibit the enzyme by at least 1000-fold.
• Ionic Strength: The enzyme’s activity is highest in buffers containing Mg2+ and Ca2+ without other salts. Increasing the concentration of salts like NaCl or KCl can reduce its activity.
• Recommended Buffer: A typical 10X DNase I buffer consists of 100 mM Tris pH 7.5, 25 mM MgCl2​, and 5 mM CaCl2​.

Discover high-quality DNase I reaction buffer >

Removing DNase I after digestion

Effectively removing DNase I after the reaction is critical, especially if the RNA will be used for cDNA synthesis.

• Heat Inactivation: Heating at 75°C for 10 minutes is a common method, but heating RNA in the presence of the divalent cations found in DNase I buffer can cause enzyme-independent RNA degradation.
• Specialized Reagents: Commercial kits are available that contain reagents to sequester and inactivate DNase I and cations, which can then be easily removed by centrifugation.
• Troubleshooting: It is important to note that DNase I can be a “sticky” enzyme, with as much as 50% of its activity adhering to the walls of certain microfuge tubes and plates. Using RNase-free microfuge tubes is recommended for best results.

In conclusion, Deoxyribonuclease I is an indispensable enzymatic reagent in molecular biology underpinning the success of a wide range of critical experimental workflows. Its judicious application is essential for obtaining high-purity nucleic acid preparations, mapping genomic regulatory elements, characterizing molecular interactions, and optimizing cellular analyses. A thorough understanding of its properties and applications is therefore vital for any researcher striving for precision and accuracy in their investigations.

## 

For Research Use Only. Not for use in diagnostic procedures.   

© 2025 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.