Deoxyribonuclease I (DNase I) is an endonuclease, its application is not only to maintain RNA integrity but also for DNA footprint analysis, generation of random DNA libraries, reduction of stickiness in cell lysates or protein extracts, etc. In a word, DNase I can be used in almost any application that requires enzyme cleavages of DNA. The following is a detailed introduction to DNase I and its specific application.

1. What is DNase I?
2. DNase I for preparation of DNA-free RNA extraction
3. DNase I for in vitro transcription to remove template DNA
4. DNase I for rRNA removal
5. DNase I for DNA labeling
6. Other applications
7. DNase I product selection guide

1. What is DNase I?

Deoxyribonuclease I (DNase I) is a non-specific endonuclease that can digest single - or double-stranded DNA, which is present in different tissues and body fluids. It can hydrolyze phosphodiester bonds to produce mono- and oligodeoxynucleotides containing a 5'-phosphate group and a 3'-OH group. The optimal working pH range of DNase I is 7-8, its activity depends on Ca2+, and can be activated by divalent metal ions such as Mn2+, Mg2+, Zn2+, etc. In the presence of Mg2+, DNase I randomly shear any site of double-stranded DNA; in the presence of Mn2+, DNase I can shear DNA double-stranded at the same site to form a blunt end or a 1-2 nucleotide sticky ends overhangs.

Figure 1. Schematic diagram of the cleavage of dsDNA by DNase I in the presence of Mg2+ and Mn2+.

Although DNase I cleavages are generally considered to be non-specific cleavages, DNase I is more likely to act on certain sequence fragments, such as the minor groove region, and is more prone to cleavages of purine-pyrimidine sequences. However, when DNase I act on heterogeneous dsDNA, all four bases will be cleaved, and the effect on a specific base will not be more than 3 times greater than that of other bases.

2. DNase I for preparation of DNA-free RNA extraction

In biological experiments, the first step is to prepare nucleic acid to study various functions of RNA. However, since DNA and RNA are often released together during the cell lysis process, the interference in the two cannot be avoided no matter what extraction solution is used, so specific enzymes need to be used to remove the interference. For high-quality RNA extraction, DNase I is used to removing residual DNA from the sample.

DNase I can degrade double-stranded and single-stranded DNA to oligonucleotides and single nucleotides, and the DNA in the RNA preparation product can be effectively degraded. DNase I is then inactivated by heating with a stop buffer. During the heating process, the hairpin structure of the RNA molecule can be opened, which facilitates the direct entry of the RNA into the reverse transcription process.
The quality of RNA will directly affect the experimental data to a large extent. In general, gDNA residues cannot be completely avoided during RNA extraction, therefore, it is generally recommended to treat RNA samples with DNase I to digest residual gDNA before challenging downstream applications (e.g. mRNA expression analysis, transcriptome analysis, etc.). The step of digesting gDNA can be performed during RNA extraction, after RNA extraction, or before RNA reverse transcription. According to the product positioning, the products provided by Yeasen are as follows:

Table 1: List of products related to DNA removal of RNA extraction or before reverse transcription

3. DNase I for in vitro transcription to remove template DNA

In vitro transcription (IVT) mainly uses DNA as a template, plus corresponding substrates and buffers to obtain RNA through in vitro transcription. In in vitro transcription experiments, RNA polymerases such as T7, T3, and SP6 are commonly used for RNA synthesis. The synthesized RNA may have DNA residues. Eliminating the DNA residues is beneficial to the development of downstream experiments. For example, in the mRNA vaccine development stage, the removal of residues is a critical step, which can reduce the difficulty of downstream purification and increase the purity of the product. The DNA template is typically removed using Recombinant DNase I (RNase-free). According to the mRNA synthesis process, the products provided by Yeasen are as follows: