DNA ligase: Unit 2 rDNA, PCR Notes Pharmaceutical Biotechnology
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It is the second type of enzyme involved in r-DNA experiments.
DNA ligase is an important cellular enzyme naturally present in micro-organisms.
Its function is to repair different broken bonds i.e. C-C, C-N, N-Metal, etc. that may occur at random or as a consequence of DNA replication or recombination.
In genetic engineering, it is used to seal discontinuities in the sugar-phosphate chains that arise when recombinant DNA is made by joining DNA molecules from different sources.
It can therefore be called molecular glue, which is used to join the DNA strands together by catalyzing the formation of a phosphodiester bond.
This function is crucial to the success of many experiments, it also helps in rectifying errors during DNA replication. DNA ligase is therefore a key enzyme in genetic engineering.
The process of ligation should be optimized because if the gene is not inserted or ligated properly within the vector then it will not multiply in the host and desired protein will not be produced. There should not be loose ends left at the site of ligation.
The enzyme used most often in experiments is T4 DNA ligase, which is purified from E.coli cells infected with bacteriophageT4. It ligates blunt ends of DNA very efficiently.
The E. coli DNA ligase creates the phosphodiester bond by using energy gained by cleaving nicotinamide adenine dinucleotide (NAD). It does not ligate blunt-ended DNA and cannot join RNA to DNA efficiently
DNA ligase Classification
Classification is done based on EC (Enzyme Commission) numbering.
Depending on the need to join different bonds, different ligase enzymes can be used. Like for joining C-C, C-N, etc bonds.
|i. Ligases used to form carbon-oxygen bonds||( EC 6.1)|
|ii. Ligases used to form carbon-sulfur bonds||( EC 6.2)|
|iii. Ligases used to form carbon-nitrogen bonds||( EC 6.3)|
|iv. Ligases used to form carbon-carbon bonds||( EC 6.4)|
|v. Ligases used to form phosphoric ester bonds||( EC 6.5)|
|vi. Ligases used to form nitrogen-metal bonds||(EC 6.6)|
SIMPLIFIED CONCEPTS OF BIOTECHNOLOGY By, Dr. Pramod Kadu & Ms. Suchita Vishwakarma
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