1DNK: DNASE I-D(GGTATACC)2 COMPLEX |
RNA samples prepared using lysis reagents may contain small amounts of contaminating genomic DNA. Removing DNA contamination from RNA samples is a crucial step before subsequent analysis, such as reverse transcription and PCR (RT-PCR), where the presence of DNA can lead to false-positive results. The most common and effective method for this is the use of the enzyme DNase I.
DNase I is a nonspecific endonuclease that selectively cleaves DNA, both single and double-stranded, by hydrolyzing phosphodiester bonds. However, DNase I must be inactivated or removed from the reaction prior to subsequent applications.
DNase I Digestion
DNase I Treatment is the gold standard for removing DNA contamination (up to 10 micrograms per milliliter). DNase I specifically degrades DNA, but not RNA.
Protocol
Materials
Always check the appropriate Safety Data Sheets, Environmental, and Health and Safety guidelines for proper handling of equipment and hazardous materials used.
Reagents
DNase I, RNase-free
DNase I reaction buffer (10x): 100mM Tris-HCl pH7.6, 25℃, 25mM MgCl2, 5mM CaCl2.
EDTA: 25 mM
Water, RNase-free
Equipment
Microcentrifuge
Microcentrifuge tubes: 1.5 ml, RNAse-free
Water bath or heat block incubator at 37 °C and 65 °C.
Method
[1] Thaw the RNA sample on ice.
[2] Add 2 microliters of 10x DNase I reaction buffer. Add one unit of DNase I per 1 to 2 micrograms of RNA. Adjust the final volume to 20 microliters with RNase-free water. Incubate the sample for 5 to 10 minutes at 37 °C.
[3] Inactivate the DNaseI by adding 2.5 microliters of 25 mM EDTA. Incubate the sample for 5 to 10 minutes at 65 to 75 °C.
[4] Centrifuge the sample briefly to collect the contents of the tube, and chill the sample on ice.
In addition to DNase I digestion, other methods for removing DNA contamination from RNA are also available.
These are:
Treatment with Proteinase K involves adding Proteinase K to degrade the DNase I, followed by a cleanup step, such as phenol-chloroform extraction. This method can be time-consuming and may lead to sample loss.
DNase removal reagents or columns, now available as commercial kits, include a reagent or spin column that binds explicitly and removes DNase I, offering a convenient and effective solution.
RNA isolation kits with integrated DNA removal include modern RNA extraction kits that incorporate a DNA removal step directly into their protocol.
On-column digestion kits often include an "on-column" DNase digestion step, where the RNA is bound to a column and then treated with DNase I before being washed and eluted. On-column digestions are an efficient method for removing DNA while minimizing sample handling and potential degradation.
Genomic DNA eliminator column kits, such as those from Qiagen, feature a specialized column that binds genomic DNA, allowing RNA to pass through.
Less common methods for the removal of DNA contamination are:
Lithium chloride (LiCl) precipitation relies on the fact that RNA is insoluble in high concentrations of LiCl, while DNA and other contaminants remain in solution.
Salt fractionation works similarly to LiCl precipitation; high concentrations of salts, such as NaCl or LiCl, selectively precipitate RNA, leaving the DNA in solution.
Acid Phenol-Chloroform Extraction is a classic method that separates RNA from DNA and proteins based on their solubility in different phases. The RNA remains in the aqueous phase, while DNA partitions to the interface or organic phase.
Please Note: Before attempting to remove DNA, it's essential to check for its presence. A standard method for doing this is by performing a control PCR reaction without the reverse transcriptase enzyme (a "-RT" control). The amplification of a product in the absence of reverse transcriptase indicates that a DNA contamination is present in the RNA sample.
Always follow the manufacturer's instructions for any kits or reagents you are using, as the specific protocols and incubation times can vary. Maintaining an RNase-free workspace, complete with dedicated equipment and reagents, is also essential for all RNA work.
References
1DNK: THE X-RAY STRUCTURE OF THE DNASE I-D(GGTATACC)2 COMPLEX AT 2.3 ANGSTROMS RESOLUTION
Weston SA, Lahm A, Suck D. X-ray structure of the DNase I-d(GGTATACC)2 complex at 2.3 A resolution. J Mol Biol. 1992 Aug 20;226(4):1237-56. [PubMed]
Green and Sambrook. Molecular Cloning. I 4th edition, 2012, pp 375-376, Cold Spring Harbor Laboratory Press.
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