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Use of Restriction Endonucleases and Ligase in Genetic Engineering
Recombinant DNA technology, gene cloning , genetic engineering, gene modification, gene splicing, or gene manipulation is the directed and deliberate manipulation of genetic make up of a cell or organism in a predetermined way of producing a genetically altered cell or organism. or otherwise, it is the manipulation of genes in desired manner under controlled conditions for the genetic modification of living systems.
- Gene Cloning Vectors
Important features of gene cloning vectors
- A donor DNA, that contains the desired DNA.
- Gene cloning vector
- A host cell (Bacteruim: E. coli)
- Different kinds of enzymes
Steps in Recombinant DNA technology or rDNA technology
Major Enzymes involved in Genetic Engineering
Three types of enzymes involved in Genetic Engineering, namely:
- Restriction Enzymes
- DNA modifying enzymes
- DNA Ligases
Restriction enzymes serve as "cutting enzymes" or "molecular scissors" . Restriction endonucleases are site specific bacterial endonucleases, which can recognize specific sequences of foreign DNA and cleave the DNA into reproducible fragments. The sequences, recognized by them are called recognition sites or recognition sequences. For the pioneering studies on restriction endonucleases William Arber, Smith and Daniel Nathans got Nobel Prize in Physiology and Medicine (1978).
Example of Restriction Enzymes: Sources and Restriction Sequences
I. Generating Cohesive Ends
Escherichia coli Ry 13
Bacillus amyloliquefaciens H
II. Generating Blunt Ends
How to name restriction enzymes?
Types of Restriction Enzymes
More than 500 kinds of restriction enzymes (RE) have been identified so far. Three major types of RE are
- Type I: Restriction enzymes make cuts at random points outside the recognition site.
- Type II: Widely used in gene cloning. Enzymes make cuts only at specific points within the recognition site.
- Type III : Restriction enzymes consists of multiple sub units. They are similar to type I enzymes
In genetic engineering, DNA Ligases serve as the key enzymes for joining DNA fragments to produce recombinant DNA.
DNA Ligases or Polynucleotide ligases are the "joining enzymes", which mediate the sealing of exposed nicks (gaps or single stranded breaks) in the backbone of the the DNA duplex. In other words, they serve as molecular glue used to stick together fragments of DNA.
DNA ligases require ATP or NAD for their activity. They accomplish this through ester bonding. DNA ligases repair broken phosphodiester bonds.