Product Class: Other

HiFi Taq DNA Ligase
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This product is available in a glycerol-free format.

 

Product Introduction

HiFi Taq DNA Ligase will ligate this substrate:

Nicked DNA/RNA

Nicked substrate for ligase selection:5´ [ straight line ] OH p [ straight line ] 3´3´ [ straight line ] 5´

Ligase substrate key with [ black straight line ] DNA and [ red wavy line ] RNA

An optimized blend of a thermostable DNA ligase and a proprietary additive, HiFi Taq DNA Ligase efficiently seals nicks in DNA with unmatched fidelity.

  • Reduces errors for ligation-based molecular diagnostic techniques (e.g., LCR, LDR)
  • Remains active after multiple thermal cycles
  • Exhibits increased discrimination between correct and mismatched base pairs at either side of ligation junctions, making the enzyme suitable for the detection of either 3 ́ or 5 ́ mismatches (vs. other thermostable ligases which are efficient for 3 ́ mismatches only).
  • Free Thermostable Ligase ReactionTemperature Calculator tool to estimate appropriate incubation temperature
  • This product can be purchased in larger volumes. Submit an inquiry to find out more.
  • Not sure which ligase to choose? Refer to our DNA and RNA Ligase Properties Chart or DNA Ligase Selection Chart
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Catalog # Size Concentration
M0647S 50 reactions

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Product Information

Description

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An optimized blend of a thermostable DNA Ligase and a proprietary additive, HiFi Taq DNA Ligase efficiently seals nicks in DNA with unmatched high fidelity. The formation of a phosphodiester bond between juxtaposed 5´ phosphate and 3´ hydroxyl termini of two adjacent oligonucleotides that are hybridized to a complementary target DNA is enhanced in the improved reaction buffer and mismatch ligation is dramatically reduced (1). The improved formulation allows higher resolution discrimination between ligation donors and acceptors, enabling precise detection of SNPs and other allele variants. HiFi Taq DNA Ligase is active at elevated temperatures (37–75°C) (2,3).

Please note that HiFi Taq DNA Ligase is intended for use in molecular diagnostics applications that depend on high fidelity nick ligation. It is not a substitute for T4 DNA ligase and is not suitable for cloning applications or adapter ligation/NGS library prep.

Figure 1: HiFi Taq DNA Ligase displays increased fidelity

Diagram of multiplexed substrate pools and ligase fidelity matrices for various ligases

(A) Schematic of multiplexed substrate pools. Each substrate pool contained a single splint with a defined NN at the ligation junction (e.g., AA, AC, AG…) along with all four upstream probes and all four FAM-labeled downstream probes. Each probe that encodes the base at the ligation junction is of unique length allowing for separation and analysis by capillary electrophoresis. A total of 16 substrate pools were prepared, one for each unique splint. (B) Comparison of the ligation fidelity of Ampligase (Epicentre), Taq DNA Ligase and HiFi Taq DNA Ligase. Fidelity measurements were performed using 1 μl of ligase in a 50 μl reaction mixture in the supplied buffers at 1X concentration. Reactions were incubated 30 min at 55°C, using multiplexed substrate pools as outlined in (A). Rows represent a single template sequence, while columns indicate a particular ligation product resulting from a specific pair of probes ligating with the indicated bases at the ligation junction. A dot indicates detection of a product (see legend above). The diagonal from the top left to the bottom right represents Watson-Crick ligation products; all other spaces indicate mismatch ligation products. While Taq DNA Ligase and Ampligase perform similarly under these conditions, with a range of mismatch products detectable, HiFi Taq DNA Ligase shows dramatically fewer mismatch products while maintaining high yields (image adapted from Reference 1).


Figure 2: HiFi Taq DNA Ligase exhibits increased discrimination at both sides of the ligation junction

Bar graph depicting ligation for 3´ mismatches and 5´ mismatches for HiFi Taq DNA Ligase as compared to other DNA ligases

Oligonucleotide probes targeting a 35 bp region of the λ integrase gene were incubated with 16.7 fmol of λ genomic DNA. Ligation products formed were detected by qPCR using SYBR® Green. HiFi Taq DNA Ligase (NEB #M0647) displays increased fidelity over Taq DNA Ligase (NEB #M0208) and Ampligase, showing a larger ΔCt value between probes fully complementary to the target sequence and those with a mismatched base pair at the ligation junction.


Figure 3: HiFi Taq DNA Ligase exhibits increased thermostability

Bar graph depicting relative activity and thermostability of HiFi Taq DNA Ligase as compared to Ampligase®

HiFi Taq DNA Ligase and Ampligase® (1 µl enzyme in a 50 µl reaction) were cycled (80°C for 90 seconds/94°C for 10 seconds) up to 100 times in their respective 1X reaction buffer. Ligase activity was assayed using a FAM-labeled nicked dsDNA substrate detected by capillary electrophoresis.


Figure 4: HiFi Taq DNA Ligase displays 2-fold reduction in mismatch ligation as compared to Ampligase

Ligation comparison of HiFi Taq DNA Ligase as compared to Taq DNA Ligase and Ampligase®

The C:G Watson-Crick base pair between the upstream probe 3´-terminal base and the complementary strand makes this a particularly difficult junction for a ligase to discriminate against mismatch ligation products (1).

Product Source

Purified from an E. coli strain containing the cloned ligase gene from Thermus thermophilus (Tth) HB8 (4,5,7).
This product is related to the following categories:
DNA Ligases
This product can be used in the following applications:
Non-Cloning Ligation,
DNA Ligation

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • NEB # Stored at (°C)
    M0647S -20
    Component Name Component # Stored at (°C) Amount Concentration
    HiFi Taq DNA Ligase M0647SVIAL -20 1 x 0.05 ml 50 reactions
    HiFi Taq DNA Ligase Reaction Buffer B0647SVIAL -20 1 x 0.5 ml 10 X

Properties & Usage

Reaction Conditions

1X HiFi Taq DNA Ligase Reaction Buffer

1X HiFi Taq DNA Ligase Reaction Buffer
20 mM Tris-HCl
150 mM KCl
10 mM MgCl2
10 mM DTT
1 mM NAD
0.1% Triton® X-100
(pH 8.5 @ 25°C)

Storage Buffer

10 mM Tris-HCl
100 mM KCl
1 mM DTT
0.1 mM EDTA
0.1% Triton® X-100
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

No

Application Features

Allele-specific gene detection using ligase-dependent methods including the Ligase Detection Reaction (LDR) and Ligase Chain Reaction (LCR) (2,4). Ligation of padlock probes (5).

Can be used in the Sidewinder DNA assembly technique (6).

Product Notes

  1. Reaction Conditions: Reactions with HiFi Taq DNA Ligase should be performed between 37–75°C. The optimal ligation incubation temperature for a given set of probes is typically within 5°C of the Tm of the probes annealing region and must be determined empirically for the best balance of activity and fidelity in your application. Use of the Thermostable Ligase Reaction Temp Calculator is highly recommended for selection of an incubation temperature. For a typical LDR-type assay, reaction time should be in the range of 10–60 minutes, with 15 minutes being ideal for many applications. For assays requiring denaturation/annealing/ligation cycling, we suggest denaturation @ 95°C for 30 s – 1 min, followed by annealing/ligation for 1 – 5 min at the optimized ligation temperature for your probe set.
  2. 1X HiFi Taq DNA Ligase Reaction Buffer requires NAD+ as a cofactor. NAD+ is supplied in the 10X HiFi Taq DNA Ligase Reaction Buffer. While 10X HiFi Taq DNA Ligase Buffer is stable at -20°C for 3 years, the buffer should be stored at -80°C to further extend the half-life of the NAD+ cofactor.
  3. To dilute HiFi Taq DNA Ligase that will subsequently be stored at -20°C, 50% glycerol storage buffer (Diluent Buffer A, NEB #B8001) can be used, however, the shelf life of HiFi Taq DNA Ligase diluted in this buffer will be reduced. To dilute for immediate use, 1X HiFi Taq DNA Ligase Reaction Buffer can be used.
  4. In order to achieve the highest fidelity possible with HiFi Taq DNA Ligase, we recommend using HiFi Taq DNA Ligase Reaction Buffer, however, HiFi Taq DNA Ligase can be used in Taq DNA Ligase Reaction Buffer with reduced activity and fidelity. Conversely, the use of standard Taq DNA Ligase in HiFi Taq DNA Ligase Reaction Buffer will boost the fidelity of that enzyme. HiFi Taq DNA Ligase can be used in a variety of polymerase buffers when supplemented with 1 mM NAD with reduced activity and modestly reduced fidelity.
  5. When visualizing ligation reactions by gel, some DNA sequences may exhibit a gel shift due to binding of protein components. This protein binding does not interfere with most downstream methods including amplification. We recommend pre-treatment with Proteinase K (1 μl in a typical 50 μl ligation reaction) for 30 min at 37°C before loading on a gel to remove this shift.
  6. The molar concentration of the ligase is 44 nM.

References

  1. Barany, F. (1991). The Ligase Chain Reaction in a PCR World. 5-16.
  2. Nilsson, M., H. Malmgren, M. Samiotaki, M. Kwiatkowski, B. P. Chowdhary and U. Landegren (1994). Padlock probes: circularizing oligonucleotides for localized DNA detection.. Science. 265(5181), 2085-2088.
  3. Robinson, N.E., Zhang, W., Ghosh, R. et al. (2026). Nature.
  4. Takahashi, M. et al. (1984). J. Biol. Chem. 259, 10041-10047.
  5. Barany, F. (1991). Proc. Natl. Acad. Sci. USA. 88, 189-193.
  6. Barany, F. and Gelfand, D.H. (1991). Gene. 109, 1-11.
  7. Lohman, G. et al. (2016). Nucleic Acids Res. PubMedID: 26365241

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. What applications are HiFi Taq DNA Ligase best used for?
  2. Can HiFi Taq DNA Ligase buffer be used with other DNA ligases?
  3. Can HiFi Taq DNA Ligase be used in other buffers?  
  4. What is the optimal reaction temperature for HiFi Taq DNA Ligase?
  5. How much HiFi Taq DNA Ligase is required for a reaction?
  6. How many temperature cycles will HiFi Taq DNA Ligase survive?
  7. Can HiFi Taq DNA Ligase be used for cloning?
  8. Does HiFi Taq DNA Ligase require NAD+?
  9. What is the stability of HiFi Taq DNA Ligase at 95°C?
  10. What is the stability of HiFi Taq DNA Ligase at room temperature?
  11. Why is the HiFi Taq DNA Ligase Buffer brown?
  12. Do thermostable DNA ligases (such as Taq DNA Ligase, 9°N DNA Ligase, and HiFi Taq DNA Ligase) ligate sticky ends?
  13. Does HiFi Taq DNA Ligase have activity on substrates containing RNA?
  14. What is LDR and how does it differ from LCR?
  15. What is LCR and which enzymes do you recommend?
  16. How can I design probes for LDR or LCR to maximize specificity?

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.

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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