- Degradation of DNA template in transcription reactions
- Removal of contaminating genomic DNA from RNA samples
- DNase I footprinting
- Nick Translation
Deoxyribonuclease I (DNase I) from beef pancreas is an endonuclease (glycoprotein), which preferentially cleaves the phosphodiester bond in the DNA behind pyrimidine nucleotides. This results in
a polynucleotide with a 5'-phosphate and a free OH-group in position 3'. DNase I cleaves single-stranded and double-stranded DNA as well as chromatin. The specificity of the enzyme reaction
(single-strand-'Nicks' versus double-strand breaks) is determined by the ions available.
In the presence of Mg2+ single-strand nicks are generated and in the presence of Mn2+ double-strand breaks. The pH-optimum of DNase I is 7.8 and it is activated by divalent cations. Maximum
activation requires the presence of Mg2+ and additional Ca2+.
Calcium ions (5 mM) protect DNase I from proteolytic digest. Inhibition is achieved by citrate, if activation is done by magnesium, but not if manganese has been the activator. Besides it is
inhibited by chelators such as EDTA and SDS or β-mercaptoethanol.
The enzyme is used in molecular biology techniques like digestion of DNA, in the RNA purification (ref. 2 Suppl. 1 pp. 4.1.4) or generating "random nicks" for "nick translation" (ref. 2 Suppl. 9
pp. 3.5.4-6) or 'footprint'-assays (ref. 2 Suppl. 7 chapter 12.4) or investigations on chromatin (ref. 2 Suppl. 48 chapter 21.4.1).
Unit definition: One unit is defined as that amount of enzyme which causes an increase of absorbance at 260 nm of 0.001 per minute at 25°C based on the method of Kunitz.
DNase I is readily soluble in e. g. 0.15 M sodium chloride or in reaction buffer (e. g. 50 mM Tris · HCl, pH 7.5; 10 mM MgCl2 (single-strand 'nicks') and 10 mM MnCl2 (double-strand breaks),
respectively; 50 μg/ml BSA
For storage dissolve DNase I in 50 % glycerol (w/v); 20 mM Tris · HCl, pH 7.5; 1 mM MgCl2. For stability reasons the concentrations should be at least 1 mg/ml (The maximum solubility is 10
This solution is stable for more than one year (ref. 2 Suppl. 8 page 3.12.5). The lyophilized form is stable for 2 - 5 years if kept at +4°C. If a solution is protease-free, DNase I will not
loose significant activity at pH 5 - 7 and 62°C for 5 hours. The enzyme may be heat-inactivated (10 minutes at 99°C).
RNase-free DNase I: Dissolve DNase I at 1 mg/ml in 0.1 M iodoacetic acid plus 0.15 M sodium acetate at a final pH of 5.3. The solution is then heated 40 minutes at 55°C and
cooled. Finally, 1 M CaCl2 is added to the solution to 5 mM. Store frozen in small aliquots.
(taken from Applichem)