Product Class: Other

Duplex DNase
neb31 cloned at NEB recombinant engineered Yes


Catalog #M7635

Product Introduction

M7635 Mechanism
  • DNA-specific endonuclease
  • Specifically degrades dsDNA in the presence of ssDNA
  • Cleaves DNA strand of DNA:RNA hybrid duplex
  • Products are short oligos or ssRNA (if cleaving the DNA strand of DNA:RNA hybrid)

    Duplex DNase is ideal for:
  • Removal of contaminating gDNA prior to RT-qPCR
  • Removal of contaminating DNA from RNA or protein preps
  • PCR decontamination
  • Consider DNase I (RNase-free) or DNaseI-XT for IVT applications

Product Information

Description

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

M7635 Degradation

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


M7635 dsDNA vs ssDNA

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

M7635 Hybrid Degradation

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

M7735 Degradation Analysis

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:
Exonucleases and Non-specific Endonucleases Products
This product can be used in the following applications:
cDNA Synthesis

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme required to release 3 pmol of FAM from a 35-mer FAM-BHQ1 labeled hairpin oligonucleotide in 1 minute at 30°C in a 50 µl reaction volume.

Reaction Conditions

1X NEBuffer™ r2.1

1X NEBuffer™ r2.1
50 mM NaCl
10 mM Tris-HCl
10 mM MgCl2
100 µg/ml Recombinant Albumin
(pH 7.9 @ 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+.

Protocols, Manuals & Usage

Protocols

  1. A Typical Duplex DNase Reaction Protocol (NEB #M7635)

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. Can Duplex DNase be heat-inactivated?
  2. Is Duplex DNase inhibited by salt?
  3. At what temperature should I use Duplex DNase?
  4. What is the minimal length of duplex DNA required for Duplex DNase to cleave?
  5.  Does Duplex DNase require divalent cations?
  6. What are the optimal reaction conditions for Duplex DNase?
  7. How does the unit definition for Duplex DNase compare to other duplex-specific DNases?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specification Change Notifications

Effective  February 21, 2024, unit definition and buffer changed.

Specifications

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]

Certificate Of Analysis

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]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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