Duplex DNase is an engineered double-strand-specific DNA endonuclease that preferentially degrades double-stranded DNA (dsDNA) over single-stranded DNA (ssDNA) or RNA. It will also cleave the DNA strand of a DNA:RNA hybrid duplex.
Figure 1: Duplex DNase preferentially degrades dsDNA over ssDNA
Activity on a dsDNA substrate (35 nt dsDNA hairpin) or ssDNA substrate (15 nt ssDNA) was measured by monitoring the increase in fluorescence when the substrate is degraded by Duplex DNase. FLUOR = Fluorophore; QUENCH = Quencher
Figure 2: Duplex DNase is more specific for dsDNA than DNase I
Activity of Duplex DNase (NEB #M7635) or DNase I (NEB #M0303) on a dsDNA substrate (35 nt dsDNA hairpin) or ssDNA substrate (15 nt ssDNA) was measured by monitoring the increase in fluorescence when the substrate is degraded by either enzyme in their respective reaction buffers at 30°C. Duplex DNase is > 5-fold more specific than DNase I is for dsDNA. The ratio of activity on double-stranded vs. single-stranded substrates demonstrates the increased specificity that Duplex DNase shows for dsDNA over ssDNA as compared to DNase I.
Figure 3: Duplex DNase degrades the DNA strand of a DNA:RNA hybrid
An in vitro transcription reaction (50 μl) was treated with 2 U DNase I-XT for 15 minutes at 37°C to digest template DNA. The RNA was purified using the Monarch® RNA Cleanup Kit (500 µg, NEB #T2050) and eluted in nuclease-free water (50 μl). Varying amounts (from 106-1012 copies) of Cluc template RNA were mixed with a 5´ fluorescently-labeled/3´-quenched 22 nt complementary DNA probe (0.2 µM final concentration, Tm 61.8°C) in NEBuffer™ r1.1. Duplex DNase (2U) was added, the reaction incubated at 58°C, and the relative fluorescence was monitored every 10 seconds in a Bio-Rad® CFX Touch™ qPCR instrument. The increase in fluorescence observed over time (which is not observed in the non-template control) demonstrates the ability of Duplex DNase to cleave the fluorescently labeled DNA strand of a DNA:RNA hybrid (probe:Cluc RNA). Moreover, the cleavage of the DNA probe when hybridized to the template RNA increases over time and is dose-dependent on the amount of Cluc template RNA present. FLUOR = Fluorophore; QUENCH = Quencher
Figure 4: Duplex DNase preferentially degrades dsDNA in a mixture of dsDNA and ssDNA over a wide range of temperatures
A mixture of 20 pmol of dsDNA (60 bp oligonucleotide) and 20 pmol of ssDNA (50 nt oligonucleotide) was incubated with 2 Units of Duplex DNase for 1 minute at 25°C, 37°C, 50°C or 65°C and then run on a 4% E-gel™ EX gel (Thermo Fisher Scientific®) stained with SYBR® Gold. Lane 1: dsDNA PCR Marker (NEB #N3234), Lane 2: ssDNA 50 nt, Lane 3: dsDNA 60 bp, Lanes 4–7: 25°C, 37°C, 50°C, 65°C. Duplex DNase displays increased activity and preference for double stranded DNA with increasing temperatures. FLUOR = Fluorophore
Product Source
A His-tagged engineered Duplex DNase expressed in Pichia pastoris.
This product is related to the following categories:
The following reagents are supplied with this product:
NEB #
Component Name
Component #
Stored at (°C)
Amount
Concentration
M7635S
-20
Duplex DNase
M7635SVIAL
-20
1 x 0.075 ml
2,000 units/ml
NEBuffer™ r1.1
B6001SVIAL
-20
1 x 1.25 ml
10 X
M7635L
-20
Duplex DNase
M7635LVIAL
-20
1 x 0.375 ml
2,000 units/ml
NEBuffer™ r1.1
B6001SVIAL
-20
2 x 1.25 ml
10 X
Properties & Usage
Unit Definition
One unit is defined as the amount of enzyme required to release 50 pmol of FAM from a 35mer FAM-BHQ1 labeled hairpin oligonucleotide in 1 minute at 30°C in a 50 µl reaction volume.
Reaction Conditions
1X NEBuffer™ r1.1
1X NEBuffer™ r1.1
10 mM Bis-Tris-Propane-HCl
10 mM MgCl2
100 µg/ml Recombinant Albumin
(pH 7 @ 25°C)
Usage Concentration
2,000 units/ml
Storage Buffer
10 mM Tris-HCl
50 mM KCl
50% Glycerol
pH 7.4 @ 25°C
Heat Inactivation
75°C for 10 minutes in the presence of 1 mM DTT. If the sample contains RNA, we recommend adding EDTA (10 mM final concentration) and DTT (1 mM final concentration), prior to heat inactivation. RNA may degrade at temperatures >65°C in the presence of divalent metals such as Mg2+.
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Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
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