Deoxyribonuclease I, more commonly known as DNase I, is an enzyme that digests DNA. It is an endonuclease, which specializes in cleaving the bonds within a nucleic acid chain. In research laboratories, DNase I is a tool for removing unwanted DNA from experimental samples. Roche is a manufacturer of high-quality DNase I used in procedures where DNA could interfere with results, ensuring the purity of molecules like RNA or proteins.
Enzymatic Function and Mechanism
DNase I functions by hydrolyzing the phosphodiester bonds that form the backbone of a DNA molecule. This action breaks the DNA strand into smaller pieces, effectively digesting it. The enzyme is not specific to a particular DNA sequence, meaning it cleaves the DNA at random locations. It is capable of acting on both single-stranded and double-stranded DNA molecules.
The activity of DNase I is dependent on the presence of divalent cations, which act as cofactors. The specific cation present influences how the enzyme cuts the DNA. When magnesium ions (Mg2+) are present, DNase I nicks one strand of a double-stranded DNA helix at independent, random sites. This process leaves the other strand intact at that location.
In contrast, when manganese ions (Mn2+) are the primary cofactor, the enzyme’s behavior changes. With manganese, DNase I can cleave both strands of the DNA molecule, often at sites that are close to one another. This results in double-stranded breaks, cutting the DNA into smaller fragments more directly.
Primary Applications in Molecular Biology
gDNA Removal from RNA Samples
One of the most common applications for Roche DNase I is the removal of contaminating genomic DNA (gDNA) from RNA preparations. When researchers isolate RNA from cells, small amounts of the cell’s gDNA are often carried over. This contamination is problematic for sensitive downstream applications that specifically analyze RNA, such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
During RT-qPCR, reverse transcriptase is used to create a DNA copy (cDNA) of the RNA, which is then amplified. If gDNA is present in the sample, it can also be amplified along with the cDNA, leading to inaccurate measurements of gene expression. Treating the RNA sample with DNase I before the reverse transcription step ensures that contaminating gDNA is digested, so only the RNA-derived signal is detected.
Cell Culture Maintenance
In cell culture, DNase I helps maintain healthy cell populations. When cells die in a culture, they lyse and release their contents, including long strands of DNA. This released DNA is sticky and can form web-like networks that cause living cells to clump together, which can negatively affect cell viability. Adding DNase I to the cell culture medium, particularly during cell harvesting, breaks down the extracellular DNA, preventing clumps and making it easier to create a uniform single-cell suspension.
Tissue Digestion
Creating single-cell suspensions from solid tissues is a necessary first step for many analyses, including cell sorting and single-cell sequencing. This process uses proteolytic enzymes, such as trypsin or collagenase, to break down the extracellular matrix. As cells are liberated from the tissue, some will die and release their DNA. Similar to its function in cell culture, this released DNA creates a sticky mesh that can trap the newly freed cells. DNase I is included in the tissue digestion buffer to digest these DNA nets, improving the yield of the single-cell preparation.
Usage Guidelines and Inactivation
The enzyme is often supplied as a lyophilized (freeze-dried) powder, which must be reconstituted before use. Water is the recommended solvent for reconstitution, and it’s advised to dissolve the enzyme to a concentration of at least 1 mg/mL. During this process, it is important not to vortex the solution, as this can denature the enzyme.
Once reconstituted, the DNase I solution should be stored correctly to maintain its activity. For short-term storage, the solution can be kept at 2 to 8°C. For long-term storage, it is best to store the enzyme at -20°C in a buffer containing glycerol to prevent freezing. Aliquoting the enzyme into single-use volumes avoids repeated freeze-thaw cycles that can reduce its activity.
After the DNase I has performed its function of digesting DNA, it is often necessary to stop its activity completely. This inactivation prevents the enzyme from degrading any DNA that may be part of subsequent experimental steps, such as cDNA synthesized from an RNA template. There are two primary methods for inactivating DNase I.
The first method is heat inactivation. This involves heating the sample to a specific temperature, around 75°C for 10-15 minutes. The heat causes the DNase I protein to denature, which permanently destroys its enzymatic function.
The second method involves chelation, which uses a molecule called EDTA (ethylenediaminetetraacetic acid). EDTA strongly binds to the divalent cations (like Mg2+) that DNase I requires to function. By removing these essential cofactors from the solution, EDTA effectively stops the enzyme’s activity.
Roche DNase I Grades and Purity Standards
Roche offers different grades of DNase I, allowing researchers to select the product that best fits their experimental needs. A key distinction among these products is the “RNase-free” designation. For any application involving RNA, it is important to use a DNase I preparation that is free from contaminating Ribonucleases (RNases). RNases are enzymes that degrade RNA, and their presence would compromise the integrity of the sample.
The highest quality grade, often labeled as Grade I or recombinant RNase-free, is manufactured and purified to eliminate any detectable RNase activity. This is achieved through quality control testing where the enzyme is incubated with an RNA standard to confirm that no degradation occurs. Using this grade ensures that the target RNA molecules remain intact during the DNA removal step.
Roche also provides recombinant versions of DNase I. Recombinant enzymes are produced using genetic engineering techniques, by expressing the bovine DNase I gene in a host system like yeast. This production method results in a highly pure and consistent product, free from other enzymes that might be present in preparations from traditional animal sources.
The quality control standards for these premium grades also test for the absence of other contaminating enzymes, such as proteases. For applications in regulated environments, such as the manufacturing of therapeutics, Roche also offers GMP (Good Manufacturing Practice) grade DNase I, which is produced under more stringent controls.