DNase I is a versatile enzyme that nonspecifically cleaves DNA to release 5′-phosphorylated di-, tri-, and oligonucleotide products (1). A powerful research tool for DNA manipulations, DNase I is used in a range of molecularbiology applications. Some of its uses include:
1. Degradation of contaminating DNA after RNA isolation,
2. “Clean-up” of RNA prior to RT-PCR and after in vitro transcription,
3. Identification of protein binding sequences on DNA (DNase I footprinting),
4. Prevention of clumping when handling cultured cells, and
5. Creation of a fragmented library of DNA sequences for in vitro recombination reactions.
While frequently used in the laboratory, the activity of DNase I is still a mystery to many researchers. Are the “units” of one source of DNase I the same as that of another? Are calcium and magnesium ions required for activity? Will DNase I degrade DNA in DNA:RNA hybrids? Can DNase I remove 100% of DNA contamination from RNA preparations? In this article we will try to answer some of the questions surrounding this commonly used enzyme.