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.[1] It has also been used during RNA extraction from Sindbis virus.[2]
• 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+.[3] 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.[4] 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.[5] 2-Mercaptoethanol, chelators, sodium dodecyl sulfate (SDS)[6] and actin[7] are known to inhibit the enzyme activity.
• Features and Benefits: Effective for nicking DNA and removal of DNA from protein samples
• Unit Definition: 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.
• 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.