WarmStart Nt.BstNBI is a site specific nicking endonuclease cloned from Bacillus Stereothermophilus. It is formulated with a reversibly-bound aptamer, which inhibits its nicking activity at temperatures below 40°C. WarmStart Nt.BstNBI cleaves only one strand of DNA on a double-stranded DNA substrate. The nicking endonuclease catalyzes a single strand break 4 bases beyond the 3′ side of the recognition sequence.
Highlights
Nt.BstNBI catalyzes a single-stranded nick 3′ of its recognition sequence. WarmStart functionality is achieved by formulation with a DNA aptamer that inhibits enzyme activity below 40°C
Figure 1: No detectable activity of WarmStart Nt.BstNBI at 25 °C
A) Nicking activity on phage T7 DNA. 1 μg T7 phage DNA was incubated with various amounts of WarmStart Nt.BstNBI (NEB #R0725) and Nt.BstNBI (Non-WarmStart, NEB #R0607) in a 50 μl reaction in NEBuffer r3.1 for 1 hour at 25°C or 55°C. 8 units of enzyme was added and a 2-fold serial dilution was performed. As indicated with “+”, full activity is observed for both enzymes at the permissive temp (55°C) but not at the restrictive temp (25 °C) for the WarmStart version.
B) Nicking activity on a fluorescently labeled DNA duplex. 2 pmol 48-bp DNA duplex containing an internal recognition site for Nt.BstNBI was incubated with various amounts of WarmStart Nt.BstNBI (NEB #R0725) and Nt.BstNBI (Non-WarmStart, NEB #R0607) in a 20 μl reaction with NEBuffer r3.1 for 30 min at 25°C or 55°C. 10 units of enzyme was added and a 2-fold serial dilution was performed. The reactions were quenched in 20 mM EDTA, 0.1% Tween 20 and then diluted with water to the final concentration of FAM-labeled DNA duplex at 1 nM. The reactions were analyzed by capillary electrophoresis (CE) fragment analysis on an Applied Biosystems 3730xl Genetic Analyzer (96 capillary array). % Product was determined as the area of the product peak divided by the total area of all peaks in the FAM channel. The average and the standard deviation of % product (Y-axis) plotted with log phase of nicking enzyme units (X-axis) was taken from triplicate reactions.
Conclusion: The WarmStart version exhibits similar activity to the regular version at 55°C but has no detectable activity at 25°C.
Figure 2: Reaction temperature profile of WarmStart Nt.BstNBI
Nicking activity of WarmStart Nt.BstNBI (NEB #R0725) and Nt.BstNBI (Non-WarmStart NEB #R0607) on a fluorescently-labeled DNA duplex was measured at various temperatures (25°C to 80°C, 5°C intervals). Reactions containing 0.125 unit WarmStart or non- WarmStart Nt.BstNBI, 100 nM DNA duplex (5’ 6-FAM labeled) in 20 μl NEBuffer r3.1 were incubated for 30 min at various temperatures. The average and the standard deviation of % product (Y-axis) plotted with temperatures (X-axis) was taken from triplicate reactions.
Conclusion: WarmStart Nt.BstNBI shows less than 10% activity below 40°C and is undetectable below 30°C, thereby enabling reaction setup at room temperature with no unintended conversion of substrate.
Figure 3: WarmStart Nt.BstNBI facilitates SDA by allowing room temperature setup
Identical Strand Displacement Amplification (SDA) reactions were run either after incubation at 25°C for 30 minutes (to mimic room temperature reaction setup) or immediately after setup on ice, using WarmStart Nt.BstNBI (NEB #R0725) or Nt.BstNBI (Non-WarmStart, NEB #R0607). Reactions were performed in 25 μl of 1X Isothermal Amplification Buffer (NEB #B0537) with 0.5 μl LAMP Fluorescent Dye (NEB #B1700), 0.4 mM dNTPs (NEB #N0447), 10 ng of template DNA (HeLa genomic DNA, 290 copies), 0.5 mM of each primer, 1 μl Bst 2.0® Polymerase (NEB #M0537S) and 1 μl WarmStart Nt.BstNBI or Nt.BstNBI. Isothermal amplification was performed at 55°C and fluorescence was monitored for 45 minutes. The time differential for the appearance of positive signal in test samples and negative controls is distinguishable only for reactions set up on ice (both versions) or at room temperature (WarmStart version only). The variation between technical replicates was low.
Conclusion: Only WarmStart Nt.BstNBI enabled successful reaction setup at room temp for SDA reactions targeting a hBRCA1 amplicon in genomic DNA.
Product Source
An E. coli strain that carries the cloned Nt.BstNBI gene from Bacillus stereothermophilus 33M (Z. Chen).
This product is related to the following categories:
10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
200 µg/ml Recombinant Albumin
50% Glycerol
pH 7.4 @ 25°C
Heat Inactivation
80°C for 20 minutes
Methylation Sensitivity
dam methylation: Not Sensitive dcm methylation: Not Sensitive
CpG Methylation: Not Sensitive
Activity at Temperature
@37°C: 0%
Advantages and Features
Features
WarmStart technology allows for room temperature setup, making applications such as Strand Displacement Amplification (SDA) user friendly.
WarmStart technology inhibits the unintentional reaction below the permissive reaction temperature, which makes experiments consistent and reproducible.
The optimal reaction temperature range is 50-60°C. Less than 10% activity is observed when the temperature is below 40°C.
When using WarmStart Nt.BstNBI with Bst 2.0 in strand displacement amplification, specific reaction conditions will vary. For best results, use 1X Isothermal Amplification Buffer.
References
Song, Q. et al. (2010). Anal.Chem. [Epub ahead of print].
Zhang, P. et al. (2010). ProteinExpr. Purif. 69, 226-234. [Epub 2009 Sep 9].
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Specifications & Change Notifications
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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.
Licenses
Nucleic acid-based aptamers for use with nicking enzymes are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use the WarmStart® Nt.BstNBI for Research Use Only (RUO). Commercial use of this product may require a license from New England Biolabs, Inc. For additional information or to inquire about commercial use, please contact busdev@neb.com.
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