DNase I Demystified
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.
Better Unit Definition
Optimal Digestion Conditions
The ionic strength of the reaction buffer is another factor that can affect DNase I activity. DNase I has optimal activity in buffers containing Mg2+ and Ca2+, but no other salts. When the salt concentration (NaCl or KCl) of the standard reaction buffer is increased from 0 to 30 mM, DNase I activity drops more than 2-fold (4). Although they contain salt, digestion of DNA in Ambion's MAXIscript™, MEGAscript™ and other transcription buffers works well since the total number of units of DNase I added to degrade the DNA template is in large excess. See the sidebar, "Treating RNA Samples with DNase I", for a suggested DNase I Digestion Buffer.
Ambion's Technical Service Department is frequently asked whether DNase I cleaves only dsDNA or whether it can also degrade single-stranded DNA (ssDNA) and the DNA in RNA-DNA hybrids. DNase I can cleave the latter 2 types of substrates, although its activity for these substrates is much reduced. For example, the specific activity of DNase I for ssDNA is about 500 times less than that for dsDNA (4). Activity on RNA-DNA hybrids is <1-2% of that for dsDNA (5). It is important to note, however, that DNase I is often used at concentrations much higher than may be necessary. For example, experiments at Ambion have shown that as much as 1 µg of 100-mer oligonucleotide can be reduced to <5-mers after a 15-min incubation with 2 U of Ambion DNase I (Cat. No. AM2222). As a result, the extent of cleavage of ssDNA and RNA:DNA hybrids will depend on the exact assay conditions.
Removing Contaminating DNA in RNA Preparations
Getting Rid of all the DNA
- Vanecko, S and Laskowski, M (1961). J Biol Chem 236: 3312-3316.
- Kunitz, M (1950) J. Gen Physiol 33: 349-362.
- Moore, S (1981) Pancreatic DNase, in: The Enzymes (P.D. Boyer, Ed.) Academic Press, New York, Chapter 15.
- Latham, G (Ambion, Inc.), unpublished data.
- Sutton, DH, Conn, GL, Brown, T, and Lane, AN (1997) Biochem J 321 (Pt 2): 481-486.
- Chomczynski, P, and Sacchi, N (1987) Anal Biochem 162: 156-159.
- Kitlinska J, and Wojcierowski, J (1995) Anal Biochem 228: 170-172.
Did You Know?
DNase I is a sticky enzyme. In some microfuge tubes and 96-well plates we have measured that as much as 50% of the input DNase activity can adhere to the container walls in just 10 minutes! For best results use Ambion's non-stick RNase-free microfuge tubes (Cat #12450) for DNase I digestions.
Getting Rid of DNase After the Reaction
DNase I treatment is easy. It's getting rid of the DNase I afterwards that can present a challenge; and effective DNase I removal is critical if the RNA will be used to synthesize cDNA. While DNase I can be removed by phenol extraction, many researchers avoid this method for fear of loss of precious RNA sample during the extraction, and because it is time consuming and requires handling phenol, a hazardous chemical. In addition, any residual phenol can inhibit cDNA synthesis. Many researchers inactivate DNase I by heat denaturation at 75ÐC for 10 min. However, this method, too, can prove deleterious for the RNA sample, since heating RNA in the presence of divalent cations, contained in DNase digestion buffer, can cause enzyme-independent degradation of the RNA. Ambion's DNA-free™ (Cat #1906) contains not only DNase I and an optimized DNase I buffer, but also a novel DNase Removal Reagent. The DNase Removal Reagent when simply added to the completed digestion, sequesters DNase I and cations, inactivating DNase activity in minutes. Simply spin to pellet the DNase Removal Reagent and proceed.
Reprinted from Ambion's TechNotes Newsletter 8:4, © 2001
10X DNase I Buffer
100 mM Tris pH 7.5
25 mM MgCl2
5 mM CaCl2
* 1 unit of DNase I is defined as the amount of enzyme that will degrade 1 µg of DNA in 10 min at 37°C.