Deoxyribonuclease I (DNase I) is an enzyme widely used in molecular biology laboratories. It breaks down DNA molecules, a process often necessary for various experimental procedures.
Understanding DNase I
DNase I is a deoxyribonuclease enzyme (EC 3.1.21.1) that cleaves phosphodiester bonds within the DNA backbone. This action produces smaller DNA fragments, typically oligonucleotides. It is a calcium-dependent enzyme, with its active site containing two calcium ions essential for its function.
DNase I is naturally found in various vertebrate organs, including the bovine pancreas, a common source for its purification. It is a glycoprotein with a molecular weight of approximately 30-32 kilodaltons. The enzyme acts on both single-stranded and double-stranded DNA, showing a preference for double-stranded DNA. Its mechanism involves a nucleophilic attack on the DNA phosphorus group by a water molecule, facilitated by specific histidine residues in its active site.
Key Applications of DNase I
DNase I serves multiple purposes in laboratory settings. A primary use is removing genomic DNA contamination from RNA samples, important for sensitive applications like reverse transcription PCR (RT-PCR) and RNA sequencing (RNA-seq). By selectively degrading DNA while leaving RNA intact, it ensures accurate downstream analysis of RNA expression.
The enzyme is also employed for fragmenting DNA into specific sizes for various assays. This includes techniques such as chromatin immunoprecipitation sequencing (ChIP-seq) and DNase I footprinting, where controlled DNA degradation helps identify protein-binding sequences on DNA. In DNA footprinting, bound proteins protect their DNA binding sites from DNase I cleavage, leaving a “footprint” that can be analyzed. Additionally, DNase I is used in preparing DNA libraries for cloning or other experimental purposes, providing a controlled method for generating random DNA fragments.
DNase I assists in cell lysis and nucleic acid extraction protocols by degrading DNA that can cause samples to become viscous. This helps release RNA or proteins more efficiently from cell lysates. It is also used for removing DNA from protein preparations, ensuring the purity of protein samples for further study.
Invitrogen’s DNase I Products
Invitrogen, a brand under Thermo Fisher Scientific, offers a range of DNase I products. Their offerings often include recombinant DNase I, produced in animal-free systems. This ensures higher purity and reproducibility compared to traditional bovine pancreas-derived enzymes, and minimizes the risk of co-purified contaminants like RNases.
A key feature of Invitrogen’s DNase I products is their RNase-free formulation, which protects RNA sample integrity during DNA removal. This is beneficial for RNA-sensitive applications like RT-PCR and RNA-seq, where trace amounts of RNase can degrade RNA. Invitrogen provides various formats, including lyophilized enzymes and solutions like TURBO DNase™.
TURBO DNase™ is an engineered variant of wild-type DNase I with enhanced catalytic efficiency and a higher affinity for DNA. This allows it to effectively remove trace quantities of DNA contamination and maintain activity in higher salt concentrations, which can inhibit conventional DNase I.
Handling and Storage Considerations
Proper handling and storage of DNase I are important for maintaining its enzymatic activity and ensuring experimental consistency. DNase I is typically supplied as a lyophilized powder or in a glycerol-containing solution. Lyophilized forms are generally stored at 2°C to 8°C, while solutions are kept at -20°C to -30°C in a non-frost-free freezer to prevent degradation.
When reconstituting lyophilized DNase I, sterile water is commonly used. The reconstituted enzyme should be stored in aliquots at -15°C to -25°C to avoid repeated freeze-thaw cycles, which can reduce activity. DNase I activity depends on divalent cations like magnesium (Mg2+) and calcium (Ca2+), often included in recommended reaction buffers. Inhibitors such as metal chelators (e.g., EDTA, EGTA), reducing agents (e.g., DTT, β-mercaptoethanol), and high ionic strength can negatively affect enzyme activity. General laboratory safety precautions, including wearing appropriate protective eyewear, clothing, and gloves, should always be followed when handling the enzyme.