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NEB/HiFi Taq DNA Ligase/50 reactions/M0647S

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货号: M0647S

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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
  (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
  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
  HiFi Taq DNA Ligase and Ampligase® (1 µl enzyme in a 50 µl reaction) were cycled (80°C for 90 seconds/94°C for 10seconds) up to 100times 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
  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 that carries the cloned ligase gene from a hyperthermophilic organism.
  This product is related to the following categories:
  DNA Ligases Products
  This product can be used in the following applications:
  Non-Cloning Ligation,
  DNA Ligation
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NEB/HiFi Taq DNA Ligase/50 reactions/M0647S

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