WarmStart technology을 적용하여 40°C 이하에서 효소 활동을 억제하는 WarmStart® Nt.BstNBI를 소개합니다.
신제품 WarmStart® Nt.BstNBI는 WarmStart technology을 적용하여 40°C 이하에서 효소 활동을 억제하여 상온 setup이 가능하며 실험을 일관되게 재현할 수 있어 Strand Displacement Amplification (SDA)에 최적화되어 있습니다. WarmStart® Nt.BstNBI는 site specific endonuclease로 인식된 single strand 3′ 서열(5’…GAGTCNNNN/ … 3’)을 끊어줍니다.
- This is a nicking endonuclease
- WarmStart technology inhibits enzyme activity below 40°C
- Generates DNA molecules that are “nicked,” rather than cleaved
- Allows for room temperature setup, facilitating SDA
- Restriction Enzyme Cut Site: GAGTC(4/-5)
| NEB # | Component Name | Amount | Concentration |
| R0725S | WarmStart® Nt.BstNBI | 1 x 0.1 ml | 10,000 units/ml |
| NEBuffer™ r3.1 | 1 x 1.25 ml | 10 X |
WarmStart® is the term we use to describe a mesophylic enzyme that is inactive at room temperature, and activated when the reaction is warmed above approximately 40°C. The WarmStart technology can be found in the following products: Bst 2.0 WarmStart DNA Polymerase (NEB# M0538), WarmStart RTx Reverse Transcriptase (NEB# M0380), Luna one-step RT-qPCR products (www.lunaqpcr.com), and WarmStart Nt.BstNBI (NEB# R0725).
WarmStart® Nt.BstNBI can be used in applications that Nt.BstNBI is used. The aptamer in WarmStart Nt.BstNBI inhibits nicking activity below 40°C, therefore the enzyme only becomes fully activated when reaction temperatures of 50-60°C are reached. This feature allows high-throughput assay assembly and room temperature setup (such as SDA). In addition, since the enzyme activity is precisely controlled, the results are consistent and variations between replicates are low.
Strand displacement amplification is an isothermal in vitro DNA amplification method that relies on a nicking enzyme to nick one strand of double-stranded DNA and a DNA polymerase with exonuclease deficiency to extend the 3′-end at the nick and displace the downstream DNA strand (GT Walker, 1992). This method can be applied to rapidly detect a target nucleic acid with high sensitivity in a point-of-care setting and has become a popular molecular diagnosis method. We show a typical SDA reaction with WarmStart Nt.BstNBI (NEB #R0725) and Bst 2.0 WarmStart DNA polymerase (NEB #M0538) to detect hBRCA1 target in HeLa genomic DNA in WarmStart Nt.BstNBI product page. To learn more and to view our strand displacement amplification and nicking enzyme amplification reaction product offerings, please visit here.
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.
