Description
Application
DNAse I is used to nick DNA, as a first step to incorporate labeled bases into DNA. The enzyme from Sigma has been used in the nuclease stock along with RNAse during cell lysate preparation of Madin-Darby canine kidney (MDCK) II cell lines. It has also been used during RNA extraction from Sindbis virus.
Deoxyribonuclease I from bovine pancreas has been used for the identification, localization and expression of two novel human genes similar to deoxyribonuclease I. Deoxyribonuclease I from bovine pancreas has also been used in a study to investigate a new approach to obtaining high-activity RNase, DNase, cholesterolesterase, and trypsin from cattle pancreas.
Used for the removal of DNA from protein samples.
Biochem/physiol Actions
DNase I is an endonuclease that acts on phosphodiester bonds adjacent to pyrimidines to produce polynucleotides with terminal 5′-phosphates. In the presence of Mg²⁺, DNAse I cleaves each strand of DNA independently and the cleavage sites are random. Both DNA strands are cleaved at approximately the same site in the presence of Mn²⁺. The pH optimum is found to be between 7 and 8. Divalent cations such as Mn²⁺, Ca²⁺, Co²⁺, and Zn²⁺ are activators of the enzyme. A concentration of 5 mM Ca²⁺ stabilizes the enzyme against proteolytic digestion. DNAse I from bovine pancreas consists of four chromatographically distinguishable components, A, B, C, and D, with their molar ratios being 4:1:1 respectively. Only minor amounts of D are found. 2-Mercaptoethanol, chelators, sodium dodecyl sulfate (SDS) and actin are known to inhibit the enzyme activity.
Features and Benefits
Effective for nicking DNA and removal of DNA from protein samples
Physical form
Contains calcium chloride
Preparation Note
The enzyme powder may be reconstituted in water or any buffer at pH 4.0-9.0, except phosphate buffer. Calcium chelators should be avoided. 10 mg/mL solution of DNAse I in 0.15 M NaCl may lose <10% of its activity when stored for a week in aliquots at −20 °C. The same solutions stored in aliquots at 2-8 °C can lose approximately 20% activity. It remains active for up to five hours at 60 °C between pH 5 and 7, and loses activity in <10 minutes at 68 °C. It loses activity at the rate of 6%/hour in acetate buffer (pH 5.0) and tris buffer ((pH 7.2) at 1 mg/mL concentration.
Analysis Note
Protein determined by biuret.
Other Notes
One Kunitz unit will produce a change in A₂₆₀ of 0.001 per minute per ml at pH 5.0 at 25 °C using DNA, Type I or III, as the substrate.
