DNase I Demystified
| 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 molecular biology 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. |
Better Unit Definition
The specific activity of a given DNase I preparation reflects the potency of the enzyme per unit mass in degrading double-stranded DNA (dsDNA). Historically, this activity has been expressed in Kunitz units (2), where 1 Kunitz unit is the amount of DNase I added to 1 mg/ml salmon sperm DNA that causes an increase of 0.001 absorbance units per min when assayed in a 0.1 M NaOAc (pH 5.0) buffer. This buffer, however, is not representative of the conditions that are typical for DNase I digestions (see "Treating RNA Samples with DNase I" at right). Consequently, Ambion offers DNase I with a unit designation that better reflects how well the enzyme will degrade DNA under standard conditions. Updated procedures measure DNase I activity using real-time fluorescence assays which allows for fast, quantitative measurements.
Optimal Digestion Conditions
Like many enzymes, DNase I activity is affected by the composition of the reaction buffer. Contrary to popular belief, DNase I is not active in buffers containing Mg2+ yet lacking Ca2+ (3). In fact, evidence in the literature suggests that what little DNase I activity is present in buffers lacking Ca2+ is due to synergistic activation by contaminating Ca2+. For this reason, the addition of EGTA at a concentration far below the Mg2+ level but above the Ca2+ level inhibits DNase I by at least 1000X and perhaps more (4). Since Ca2+ is known to bind tightly to DNase I and stabilize its active conformation, even micromolar levels of Ca2+ can act as a potent enzyme activator in the presence of Mg2+.
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.
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.
Cleavage Specificity
DNase I digestion of heterogeneous dsDNA reportedly yields dinucleotides (60%), trinucleotides (25%), and oligonucleotides. The smallest substrate for DNase I is a trinucleotide. Although DNase I is commonly perceived to cleave DNA nonspecifically, in practice it does show some sequence preference. For example, the enzyme is sensitive to the structure of the minor groove, and favors cleavage of purine-pyrimidine sequences. However, DNase I will cut at all 4 bases in heterogeneous dsDNA, and the specificity of cleavage at a given base usually does not vary more than 3-fold.
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.
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
A frequent use of DNase I is to treat RNA preparations to degrade trace to moderate amounts of genomic DNA (up to 10 µg/ml) that could otherwise result in false positive signals in subsequent RT-PCR. The amount of RNA that can be treated in a single DNase I reaction will depend on the amount of DNA contamination. DNA can be carried over from the interface of organic extractions, and when the silica matrix of solid-phase RNA purification methods is overloaded. RNA isolated from some tissues, such as spleen, kidney, or thymus, and RNA isolated from transfected cells, also tends to contain higher levels of DNA contamination. For example, RNA isolated from tumors using the method of Chomcynski and Sacchi (6) has been reported to be contaminated with 7% DNA, a level that can amount to micrograms of DNA depending on the scale of the preparation (7).
Getting Rid of all the DNA
It is probably impossible to get rid of every last strand of DNA in an RNA preparation. RT-PCR has the ability to amplify a single molecule from a complex heterogeneous mixture; in fact, RT-PCR is so sensitive that when more than 40 cycles of PCR are performed, just about any reaction will produce a band in the "minus-RT" control reaction, indicating contaminating DNA. It is important to use optimal DNase I digestion conditions and to use a cycle number relevant and reasonable for the RT-PCR experiment. To maximize chances of complete digestion, a buffer such as that described in the sidebar, "Treating RNA Samples with DNase I", should be used. In addition, it is important not to perform the digestion at too high of an RNA concentration. RNA preparations should be diluted to ~100 µg/ml for treatment. The amount of DNase I used will depend on the level of contamination (again, see the sidebar, for guidelines).
References
- 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?
Don't Lose Your DNase!
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
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
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