Ligation (molecular biology)
Ligation is the joining of two nucleotides, or two nucleic acid fragments, into a single polymeric chain through the action of an enzyme known as a ligase.
The discovery of DNA ligase dates back to 1967 and was an important event in the field of molecular biology.
Human DNA ligase abnormalities have been linked to pathological disorders characterized by immunodeficiency, radiation sensitivity, and developmental problems.
[10] The relative concentration of the DNA fragments, their length, as well as buffer conditions are also factors that can affect whether intermolecular or intramolecular reactions are favored.
The concentration of DNA can be artificially increased by adding condensing agents such as cobalt hexamine and biogenic polyamines such as spermidine, or by using crowding agents such as polyethylene glycol (PEG) which also increase the effective concentration of enzymes.
[11][12] Note however that additives such as cobalt hexamine can produce exclusively intermolecular reaction,[11] resulting in linear concatemers rather than the circular DNA more suitable for transformation of plasmid DNA, and is therefore undesirable for plasmid ligation.
[13] As is usual for an enzyme, the higher the ligase concentration, the faster is the rate of ligation.
At high concentration of monovalent cation (>200 mM) ligation can also be almost completely inhibited.
[20] For practical purposes, sticky end ligations are performed at 12-16°C, or at room temperature, or alternatively at 4°C for a longer period.
To compensate for the lower efficiency, the concentration of ligase used is higher than sticky end ligation (10x or more).
Blunt-ended PCR product normally lacks a 5'-phosphate, therefore it needs to be phosphorylated by treatment with T4 polynucleotide kinase.
Topoisomerase can be used instead of ligase for ligation, and the cloning may be done more rapidly without the need for restriction digest of the vector or insert.
For instance, Nicotinamide adenine dinucleotide (NAD+)-dependent ligase was found and isolated from bacterial organism, known as E. coli in second third of 20th century.
Examples of genes present in E. coli are LigA, which has essential functions affecting bacterial growth, and LigB.
All eukaryotes contain multiple types of DNA ligases encoded by Lig genes.
When ChVLig is the only source of ligase in the cell, it can continue to support mitotic development, and nonhomologous end joining in budding yeasts.
Nevertheless, studies of mouse embryogenesis have shown that until the middle of the growth process embryo developing without DNA ligase I.
[35] Enzymatic ligation has been used in various studies related to DNA nanostructures and lead to increase of efficiency and stability.
[2] In nanostructures architecture, molecular biology researches - ssDNA is an important application model.
T4 DNA ligase used to cyclize short ssDNA fragments, but process is complicated by formation of secondary structures.
On the other hand, Taq DNA ligase is a thermostable enzyme which can be applied at higher temperatures (45, 55 and 65 °C respectively).
Since at these temperature range secondary structures less stable it is enhance cyclization efficiency of oligonucleotides.
The kinetic, biological, and other parameters of nanostructures are influenced by presence of the secondary structures in DNA rings.
[37] Analysis of ligases activities, mutations, deficiencies widely used in drug design and biological researches to investigate diseases, pathologies developments and related rare acquired or inherited syndromes (e.g. DNA ligase IV syndrome).
[38][39][40][41] The ligation procedure is prevalent in molecular biology cloning techniques, and it has been applied to define and characterize specific nucleotide sequences in the genome using Ligase Chain Reaction (LCR) or Polymerase Chain Reaction (PCR)-based amplification of ligated probes.
