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The Taq DNA Ligase is NAD+ dependent thermostable DNA ligase from the thermophilic bacterium Thermus aquaticus. The function of this enzyme is very similar to that of Tth ligase from related thermophilic species Thermus thermophilus.
This enzyme catalyzes the formation of a phosphodiester bond at a nick junction on double-stranded DNA. This enzyme does not tolerate any mismatches around the nicked region, otherwise, even a single-nucleotide mismatch dramatically reduces the ligation activity. This unique specificity demand of thermostable ligases makes them irreplaceable in many advanced molecular techniques.
Three steps are included in the ligation cycle: enzyme adenylation, substrate adenylation and nick-closure. At the beginning, an adenosine monophosphate (AMP) group is transferred from the cofactor nicotinamide adenine dinucleotide (NAD) to a lysine residue in the adenylation motif KXDG through a phosphoamide linkage. Then, this AMP group is transferred to the 5´ phosphate at the nick through a pyrophosphate linkage to form a DNA-adenylate intermediate. At the end, a phosphodiester bond is formed to close the nick and AMP can be released.
The standard reaction buffer for Taq DNA Ligase is 200 mM Tris-HCl (pH 8.3), 250 mM KCl, 100 mM MgCl2, 5 mM NAD, and 0.1% Triton X-100. For a long term storage (>30 days), -80°C deep freezer is recommended.
Ligases from thermophilic bacteria exhibit much higher fidelity of even one up to two orders of magnitude more than bacteriophage T4 DNA Ligase. Taq DNA ligase can be used in a variety of methods (briefly described below). However, it cannot replace T4 DNA Ligase in most conventional cloning methods because of insufficient activity at low temperatures where 2- and 4-base cohesive ends form stable duplexes or blunt ends.
DNA ligases and ligase-based technologies.
Bacterial DNA ligases.
Enzymatic assembly of DNA molecules up to several hundred kilobases.
Ligase chain reaction (LCR)-overview and applications.
A review of molecular recognition technologies for detection of biological threat agents.
Direct detection of novel expanded trinucleotide repeats in the human genome.
A method for the amplification of unknown flanking DNA: targeted inverted repeat amplification.
Genetic disease detection and DNA amplification using cloned thermostable ligase.
A ligase-mediated gene detection technique.