Removes trace quantities of DNA that can plague RT-PCR
RNase-free and recombinant in origin: Purified from a source that is 107-fold lower in RNase activity than bovine pancreas
A New Hyperactive DNase with Superior Properties to Wild-type DNase I
TURBO DNase (patent pending) was developed using a protein engineering approach that introduced amino acid changes into the DNA binding pocket of wild-type DNase I. These changes lower the Km for DNA by 6-fold, compared to wild type (wt) DNase I. In addition, TURBO DNase retains at least 50% of peak activity in solutions approaching 200 mM monovalent salt, even when the DNA concentration is in the nanomolar (nM) range. Wild type DNase I in comparison, loses significant activity when the salt level reaches 20 mM NaCl. Thus, TURBO DNase is extremely efficient at removing DNA from many common molecular biology buffers, such as in vitro transcription buffer (Figure 1). In this case, TURBO DNase reduces the input 50 kb lambda DNA to digested fragments that are roughly 100 times smaller than those treated with conventional DNase I. As a result, TURBO DNase is the enzyme of choice for eliminating plasmid DNA templates from transcription reactions, particularly for the synthesis of RNA standards used in RT-PCR and generating RNA for microinjection and transfection experiments.
Figure 1. TURBO DNase Provides Superior Elimination of DNA from in vitro Transcription Reacti ons. Equivalent units of bovine pancreatic DNase I from Ambion or Promega (0.05 U), or Ambion's TURBO DNase (0.05 U) were added to a 20 l reaction containing 1 g of lamda DNA and Ambion's MEGAscript T7 In Vitro Transcription Buffer at a 1X concentration. The reaction was incubated for 13 min at 25C, quenched with EDTA, and immediately loaded on a 1% agarose gel soaked with ethidium bromide. Note that these DNase digests were performed with much less DNase I than is typically used to illustrate the dramatic difference in activity between TURBO DNase and wild-type DNase I.
High Specific Activity and Highest Quality DNase Available
One of the issues that needs to be kept in mind prior to purchasing any commercial preparation of wt DNase is the unit definition of the enzyme. Unfortunately, different vendors use widely differing assays to define unit activity, making a direct unit to unit comparison difficult. Additionally, the same number of units from different vendors can contain significantly varying amounts of active enzyme, reflecting the wide variation in specific activity for different commercial preparations of wt DNase I. Given that most wt DNase I is purified from bovine pancreas, a highly RNase-rich source (see below), one can inadvertently cause RNA degradation by using a low specific activity enzyme that is contaminated with RNase. In addition to being a hyperactive enzyme, TURBO DNase is also a very high specific activity enzyme. Thus, the same mass amount of TURBO DNase can perform significantly better than wt DNase I from other vendors (Figure 2). TURBO DNase is produced in an organism with little to no RNase contamination, further reducing the chance of RNA degradation.
Figure 2. Comparison of DNase Activity. Lambda DNA (4 g) was either left intact (Unt, 1 g), or was digested with 12 ng of TURBO DNase or DNase from Promega, Sigma, or Invitrogen, in a 60 l reaction using 1X DNase Buffer supplied by the vendor. Reactions were incubated 5 min at 37C and DNase was inactivated by 5 min, 95C heat denaturation. Aliquots (10 l) from each reaction were analyzed by 1% agarose gel and by PCR (5 l aliquot into a 25 l reaction). Corresponding "Ct values" and "fold DNA removal" are provided. The fold-removal of DNA was determined in the following manner: the Ct value for the untreated sample was subtracted from the Ct value for each treated sample and raising 2 to that number as the exponent. Thus, TURBO DNase treatment resulted in a fold-removal of 80684 (CtTD-CtUnt = 16.3. Raising 2 to 16.3 as the exponent (216.3) we obtain 80684).
We recently compared TURBO DNase to other commercially purchased preparations of wt DNase from Promega, Sigma and Invitrogen (Figure 2). Briefly, 4 g of lambda DNA was digested with 12 ng of the various DNases for 5 minutes. An aliquot from each reaction was analyzed by gel electrophoresis. A separate aliquot was used in a 40 cycle real-time PCR reaction using primers designed to amplify a specific 80 bp lambda genomic DNA amplicon. Detection was performed with a TaqMan probe. As can be seen from the gel electrophoresis data, most of the lambda DNA was digested to less than an average size of 100 bp with TURBO DNase treatment. In fact TURBO DNase treatment reduced the substrate to < 20 bp (data not shown). However, treatment with wt DNase from the other vendors left significant amounts of DNA with an average size of 100 bp.
This result was also seen using a real-time PCR assay to detect residual lambda DNA. The highest Ct values were obtained for the sample treated with TURBO DNase suggesting this sample had the least amount of amplifiable lambda DNA left in the sample. TURBO DNase showed greater than 80,000-fold removal of lambda DNA compared to the untreated sample. Treatments with wt DNase from the other vendors were 4- to 3300-fold less efficient in removing DNA.
TURBO DNase: Highly Pure, RNase-free
Wild type DNase I is typically purified from bovine pancreas, a tissue source that is extremely rich in RNases. In fact, as much as 1 mg of RNase A can be present in 1 g of pancreas tissue. Thus, many commercially available preparations of wt DNase I, even those purported to be RNase-free, can be contaminated. TURBO DNase is a recombinant protein purified from a source that is 107-fold lower in RNase activity than bovine pancreas. This special preparation protocol results in a DNase that exceeds even Ambion's stringent quality control standards for RNase contamination.
TURBO DNase is available in two convenient sizes of 1000 U and 5000 U. A specially formulated 10X Reaction Buffer is provided with the enzyme.