The enzyme DNA ligase is essential for DNA replication, recombination and for repair of genetic material in vivo. From virus and both prokaryotic and eukaryotic organisms have been isolated and characterized different DNA ligases, most recently, have been cloned and purified DNA ligases from thermophilic microorganisms. The ligases characterized to date consist of a single polypeptide chain and exhibit different activities. The ligation activity includes: 1) the binding of double-stranded nucleic acids with cohesive terminals, 2) the joining of blunt-end complex, and 3) the binding of nucleic acid fragments through gap sealing mechanism (nick -sealing).

The diagnostic technology operates almost exclusively ligase activity sealing gaps. The best-studied DNA ligases are isolated from Escherichia coli and encoded by the genome of bacteriophage T4. E. coli DNA ligase is specific for binding DNA segments that are hybridized to a DNA sequence of a mold, by contrast, T4 DNA ligase catalyzes the joining of RNA or DNA segments with any mold either RNA or DNA.

The typical substrate for sealing gaps activity comprises at least two DNA segments that are hybridized immediately adjacent positions and in the same strand of nucleic acid template (Figure-1). The 5 ‘sequence must end with a hydroxyl group (OH) 3′, whereas the sequence 3 ‘must have a phosphate (P) 5′. The enzymatic binding of the two segments of nucleic acids is carried out through a reaction that consists of three steps. Ligase-AMP complex is formed by fosfoamida connection between a lysine residue in the molecule of the enzyme and AMP derivative appropriate cofactor (either NAD or ATP). Subsequently, a connection is formed phosphodiester between AMP and phosphate complex 5 ‘probe B. Nucleophilic attack of the phosphate 5 ‘hydroxyl group by the 3′ probe A results in sealing of the chip, with release of AMP.