An engineered variant of DNase I, DNase I-XT is a salt-tolerant DNA endonuclease that nonspecifically cleaves DNA to release di-, tri- and oligonucleotide products with 5′-phosphorylated and 3′-hydroxylated ends (1,2). DNase I-XT acts on single- and double-stranded DNA, chromatin and the DNA strand of RNA:DNA hybrids. While DNase I (RNase-free)(NEB #M0303) is inhibited by salt concentrations >50 mM, DNase I-XT (NEB #M0570) exhibits optimal activity between 50-100 mM salt and retains 65% and ~40% activity in 200 and 300 mM salt, respectively. This increased salt tolerance makes DNase I-XT the preferred enzyme for DNA template removal from an in vitro transcription (IVT) reaction. Importantly, DNase I-XT is RNase-free, allowing for the complete removal of DNA from RNA preparations while maintaining RNA integrity.Figure 1: DNase I-XT efficiently degrades DNA at salt concentrations > 100 mMAn equimolar comparison of the DNase activity of DNase I (NEB #M0303) and DNase I-XT (NEB #M0570) illustrates the increased salt-tolerance of DNase I-XT as compared to DNase I. DNase activity was measured by an increase in fluorescence from a quenched 35 nt hairpin dsDNA substrate in 1X DNase I Reaction Buffer with increasing salt concentration(as indicated). While DNase I activity steadily decreases with increasing salt concentrations, DNase I-XT remains active in solutions containing up to 300 mM salt.Figure 2: DNase I-XT removes more DNA from IVT reactions and RNA preparationsIn vitro transcription reactions (20 μl) were treated with 1) no DNase I; 2) 2 U TURBO® DNase or 3) 2 U DNase I-XT for 15 minutes at 37°C. Each sample was then purified using the Monarch® RNA Cleanup Kit (500 µg, NEB #T2050) and eluted in nuclease-free water (50 μl). The level of residual DNA contamination was quantified by real-time PCR using the Luna® Universal Probe qPCR Master Mix (NEB #M3004). Average Cq (quantification cycle) values for each sample were compared to a standard curve (gray) to determine the percent of residual, PCR-amplifiable DNA. Both TURBO DNase and DNase I-XT require no dilution of the IVT reaction prior to DNase digestion, however, more DNA template is removed from an IVT reaction and undetectable by qPCR when treated with DNase I-XT.Figure 3: DNase I-XT efficiently removes residual genomic DNA from crude RNA preparationsCrude RNA samples were subjected to either: 1) No DNase treatment; 2) in-solution treatment with DNase I-XT (2 U, NEB #M0570) in DNase I-XT Reaction Buffer; 3) in-solution treatment with DNase I (2 U,NEB #M0303) in DNase I Reaction Buffer or 4) in-solution treatment with TURBO DNase (2 U, ThermoFisher) in TURBO DNase Reaction Buffer for 15 minutes at 37°C. Residual genomic DNA (gDNA) was quantified (+/– RT) by RT-qPCR using the Luna Universal One-Step RT-qPCR Kit (NEB #E3005) and human or mouse actin exonic primers. DNase effectiveness was calculated by the difference between the average Cq value -RT (amplified DNA) and the average Cq value +RT (amplified DNA and RNA).DNase I-XT removes more contaminating gDNA from crude RNA preps than either DNAse I or TURBO DNase.
Product Source
A His-tagged engineered variant of DNase I expressed in Pichia pastoris.
This product is related to the following categories:
Exonucleases and Non-specific Endonucleases Products,
DNA Modifying Enzymes & Cloning Technologies Products
This product can be used in the following applications:
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