DNA Ligation

//DNA Ligation

DNA Ligation

DNA ligation is process of joining of two nucleic acid fragments through the action of an enzyme. It is important process to create recombinant DNA , when a foreign DNA has to be insert into the plasmid. The enzyme used for the process of ligation is called Ligase.

DNA Ligase

It is specific type of enzyme that catalyze the formation of phosphodiester bond between the nucleic acids of DNA. It plays important role in repairing the breaks in DNA molecule. This is the enzyme that uses in both repairing as well as replication of DNA. The purified form  of DNA ligase is use in the formation of recombinant  DNA.

The DNA ligases are classified into two catagaries depending on the requirement of cofactor i.e. ATP-dependent or NAD-dependent. ATP-dependent ligases have been found in bacteriophages, viruses, archaea, eukaryotes and bacteria. NAD-dependent ligases exist in eubacteria and entomopoxviruses.

 E.Coli DNA ligase: This enzyme is encoded by the lig gene. DNA ligase in E.coli as well as most prokaryotes, uses energy gained by cleaving NAD to create the phosphodiester bond. It does not ligate blunt-ended DNA but require molecular crowding with polyethylene glycol and also unable to join RNA to DNA efficiently.

T4 DNA ligase: The enzyme is found in bacteriophage T4. This enzyme has ability to ligate cohesive or blunt ends of DNA, oligonucleotides, RNA and RNA-DNA hybrid. It can not ligate single stranded nucleic acids.  It uses ATP as cofactor to gain energy.

Mammilian ligases:

  1. DNA ligase I: It ligates the DNA of the lagging stand aftter Ribonuclease H has removed the RNA primer from the Okazaki fragments.
  2. DNA ligase III:  It form complexes with DNA repair protein to ligate DNA during the process of nucleotide exicion repair and recombinant fragments. Of the all known mammalian DNA ligases, only Lig III has been found to be present in mitochondria.
  3. DNA Ligase IV: It form complexes with DNA repair protein XRCC4. It catalyzes the final step in the non homologous end joining. DNA double-strand break repair pathway.
  4. DNA ligase II: it appears to be used in repair. It is formed by alternative splicing of a proteolytic fragment of DNA ligase III and does not have its own gene, therefore it is often considered to be virtually identical to DNA ligase III.

Thermostable ligases

This enzyme is isoleted from a thermophilic bacterium, As compare to conventional DNA ligases , this enzyme is more stable and active at much higher temperatures. Its half-life is 48 hours at 65°C and greater than 1 hour at 95°C. Ampligase DNA Ligase is the example of thermostable enzyme that has been shown to be active for at least 500 thermal cycles (94°C/80°C) or 16 hours of cycling. This exceptional thermostability permits extremely high hybridization stringency and ligation specificity.

Mechanism of DNA Ligase

DNA ligase form two covalent phosphodiester bonds between 3’ Hydroxyl ends of one nucleotide (“acceptor”), with the 5’phosphate end of another (“donor”). Two ATP molecules are consumed for each phosphodiester bond formed. AMP is required for the ligase reaction, which proceeds in four steps:

  1. Reorganization of activity site such as nicks in DNA segments or Okazaki fragments etc.
  2. Adenylation (addition of AMP) of a lysine residue in the active center of the enzyme, pyrophosphate is released;
  3. Transfer of the AMP to the 5′ phosphate of the so-called donor, formation of a pyrophosphate bond;
  4. Formation of a phosphodiester bond between the 5′ phosphate of the donor and the 3′ hydroxyl of the acceptor.

Sticky-end ligation

The DNA digestion of restriction enzyme leads to formation of 4-base single-stranded overhang called the sticky or cohesive end. These sticky ends ligate to other compatible ends. The sticky end ligation is performed 12-16°C, or at room temperature.

For the insertion of a DNA fragment into a plasmid vector, it is preferable to use two different restriction enzymes to digest the DNA so that different ends are generated. The two different ends can prevent the religation of the vector without any insert, and it also allows the fragment to be inserted in a directional manner.

Blunt end ligation:

Blunt end does not involves ligation of overhang. The restriction enzymes that generate blunt ends are SmaI and EcoRV. These ends are usually generated in PCR, these ends are ligated to vector generated after restriction digestion.

This ligation is less effiecient than sticky-end digestion.

Factors affecting ligation

DNA concentration

The concentration of DNA can affect the rate of ligation, As each DNA fragment has two ends, and if the ends are compatible, a DNA molecule can circularize by joining its own ends. At high DNA concentration, there is a greater chance of one end of a DNA molecule meeting the end of another DNA, thereby forming intermolecular ligation.

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 polyethyleneglycol (PEG) which also increase the effective concentration of enzymes.

Ligase concentration

The higher the ligase concentration, the faster the rate of ligation. Blunt-end ligation is much less efficient than sticky end ligation, so a higher concentration of ligase is used in blunt-end ligations.

Temperature

DNA Ligase activity is effected by melting temperature of DNA to be ligated., the optimumtempratyre for the acrivity is 37°C.

The greater the number of G and C, the higher the Tm since there are three hydrogen bonds formed between G-C base pair compared to two for A-T base pair.

Buffer composition

The ligation is also effected by ionic strength of the buffer. For example, excess amount of Na+ can cause the DNA to become more rigid and increase the likelihood of intermolecular ligation. At high concentration of monovalent cation (>200 mM) ligation can also be almost completely inhibited. The standard buffer used for ligation is designed to minimize ionic effects.

Applications:

  • DNA ligases are major tool for the production of recombinant DNA sequences. For example, DNA ligases with restriction enzymes are used to insert genes into plasmids.
  • DNA Ligase is used for cloning of full length cDNA
  • DNA origami is another application of DNA ligase. It provide enzymatic assistance to make DNA lattice structure from DNA over hangs.
  • It uses in the detection of mutation as thermostable DNA ligase retain their activity after exposure of high temperature for multiple round.
  • It is also used in DNA amplification reaction.
By |2018-05-04T05:31:45+00:00May 1st, 2018|Molecular Biology|Comments Off on DNA Ligation

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