- One unit of Taq DNA Ligase is defined as the amount of enzyme which ligates 50% of the 12-base pair cohesive ends of 1 µg of BstEII-digested λ DNA during 15 minutes at 45°C.
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.
Reaction conditions
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.
Ligase based technologies
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.
- Gibson assembly - an isothermal single-reaction method used for assembly of multiple overlapping DNA molecules, DNA ligase concerts with a 5′ exonuclease and a DNA polymerase in one reaction.
- Ligase chain reaction (LCR) or Ligation amplification reaction (LAR) – method employing thermostable ligase to discriminate DNA sequences differing in a single base pair, it is usually performed in conjunction with a primary PCR amplification.
- Ligase detection reaction (LDR) – a pair of oligonucleotid probes is annealed to the sequence of interest and joined by DNA ligase and then via repeated denaturation, annealing and ligation, the target signal is linearly amplified.
- Repeat expansion detection (RED) – method based on annealing and ligation of repeat-specific oligonucleotide probes to genomic DNA which is used as a template.
- Oligonucleotide ligation assay (OLA) – DNA ligase joins two DNA probes which are complementary to the target sequence, one of them is biotinylated for signal capture by streptavidin and the second is linked with a reporter group (32P-labeled or fluorecently labeled) for detection.
- High-fidelity gene synthesis – method based on gene synthesis from overlapping oligodeoxynucleotides containing long regions of complementary overlap, it can be improved employing thermostable ligase than T4 DNA Ligase because of increasing probability of formation of correct ligation products using higher ligation temperatures.
- Simultaneous multiple site mutagenesis – method used for proper ligation of amplified DNA fragments with point mutations introduced by mutant primers.
- Targeted inverted repeat amplification – method developed for the rapid amplification of unknown DNA flanking a known site which enables walking into uncharacterized/unknown regions of DNA.
References:
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.
