Deoxyribonucleases (DNases) are abundant in nature and can potentially degrade DNA, destroying experimental data if contained within samples for genetic analysis. Although DNases present less of an obstacle than RNases for removal from prepared samples, their detection can be a challenge using traditional methods. I have used the Ambion DNase Alert QC system and found it to be a fast and sensitive method for the detection of DNase in the lab environment including solutions, samples and materials.
The DNase Alert QC system is a fluorescent based assay that is designed to be used in conjunction with the RNase Alert QC System. A fluorescent signal is generated in samples upon cleavage of a labeled DNA substrate (provided by Integrated DNA Technologies, Inc.) indicating the presence of DNase enzyme activity. The kit can be used to evaluate almost any reagent that can not inhibit the cleavage of DNase due to reagent components or experimental conditions (such as pH extremes, high ionic strength, and/or dark colored dyes). I have used this kit in a wide range of conditions and have been satisfied with the results. Ambion claims that solid surfaces may also be tested using this system however, I have not used the kit for this purpose and thus, can not comment on this aspect.
The kit itself is easy and straightforward to use without too many steps or procedures. Kits include DNase Alert Substrate, 10X Nuclease Alert Buffer, Nuclease-Free Water, DNase I Control Enzyme, RNaseZap, TE Buffer and a small number of DNase free tubes (quantity for approximately 500 samples). The DNase Alert Substrate comes in 5 tubes, (96 samples each) which I liked because each can be resuspended with the TE Buffer individually, preventing contamination of a single large stock tube. This is not the case with the 10X NucleaseAlert Buffer, so care must be taken to prevent contamination. The DNase I comes in a fairly small quantity, but if you plan to perform a standard curve for each assay run it is readily available and fairly inexpensive from almost any reagent supplier. The remaining reagents are provided generously. I found that the Milli-Q water from my lab was actually cleaner than the Nuclease-Free water provided in the kit, however a large aliquot is included. The RNaseZap included in the kit is a nice bonus as this is a moderately expensive consumable reagent as a stand alone product.
The procedure for use of the kit is very easy. Resuspend the DNase Alert Substrate, combine with the 10X buffer and experimental samples, and incubate at 37°C for 30–60 minutes. The signal may be detected using a fluorometer with excitation and emission at 535 and 556nm, respectively. I have used 96-well plates to perform this assay in an RT-PCR instrument (Real Time format) as well as using a plate reader with starting and endpoint reads. The system is quite flexible and can be adapted to a variety of samples and instruments. The RNase Alert QC system can be used simultaneously by adding the additional RNase Alert Substrate and detecting the signal at 490 and 520nm (excitation/emission).
The features of this kit that I found to be desirable included sample flexibility, ease of use (with minimum training for even novice technicians), and compatibility with the RNase test system. In addition, the cost is not prohibitive at $480 (approximately $1 per test sample unless running quantitation standards regularly) and the RnaseZap and tubes included slightly offset the price. I would definitely recommend this assay system to anyone who is interested in an easy method for identifying sources of contamination from DNase and RNases within the lab or even as a QC system for product lines.
Daniel J. Schwartz
Scientist
Gentel Biosurfaces, Inc.
Madison, WI
| Comment on this Review |
Comments? Questions? Discuss this review of Ambion's-DNase-Alert-QC-System with the rest of the Biocompare community.
|
more articles...
