Description

• Application: DNAse I is used to nick DNA as a first step to incorporate labeled bases into DNA. DNAse I from Sigma has been used along with other enzymes for tumor harvest and dissociation, during the isolation and molecular characterization of cancer stem cells in MMTV-Wnt-1 murine breast tumors.[1]
• 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+.[2] 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.[3] 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 molar ratios being 4:1:1 respectively. Only minor amounts of D are found.[4] 2-Mercaptoethanol, chelators, sodium dodecyl sulfate (SDS)[5] and actin[6] are known to inhibit the enzyme activity.
• Features and Benefits: Efficient DNA removal: Deoxyribonuclease I from bovine pancreas is an effective endonuclease that cleaves both strands of DNA randomly, making it a reliable choice for DNA removal from protein samples. Versatile applications: This enzyme can be used to nick DNA, incorporate labeled bases into DNA, and isolate and molecularly characterize cancer stem cells in murine breast tumors. It is also useful in comparative studies of deoxyribonuclease isolated from bovine, ovine, and porcine pancreas. Stable: The enzyme remains active for up to five hours at 60 °C between pH 5 and 7, and can be stored for up to a week at −20 °C with minimal loss of activity. This stability makes it a cost-effective and convenient option.
• Unit Definition: One Kunitz unit will produce a ΔA260 of 0.001 per min per mL at pH 5.0 at 25 °C, using DNA, Type I or III as substrate.
• 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.