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Use of Restriction Endonucleases and Ligase in Genetic Engineering

Updated on February 28, 2014

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.

Basic requirements

  • 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
Steps in Recombinant DNA technology

Steps in Recombinant DNA technology or rDNA technology

Major Enzymes involved in Genetic Engineering

Three types of enzymes involved in Genetic Engineering, namely:

  1. Restriction Enzymes
  2. DNA modifying enzymes
  3. DNA Ligases

Action of EcoR1
Action of EcoR1

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

Restriction Enzyme
Source
Restriction Sequenc
I. Generating Cohesive Ends
 
 
Eco R1
Escherichia coli Ry 13
GAATTC
 
 
CTTAAG
Bam HI
Bacillus amyloliquefaciens H
GGATCC
 
 
CCTAGG
II. Generating Blunt Ends
 
 
Alu I
Arthrobacter luteus
AGCT
 
 
TCGA
Bal I
Brevibacterium albidum
TGGCCA
 
 
ACCGGT

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.

Action of DNA ligase
Action of DNA ligase

DNA ligases require ATP or NAD for their activity. They accomplish this through ester bonding. DNA ligases repair broken phosphodiester bonds.

Function of DNA Ligase in Recombinant DNA technology

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    • teaches12345 profile image

      Dianna Mendez 3 years ago

      This is very technical and scientific. Lots of information for those who seek this research. I'm not familiar with the language, but I do know it is very interesting and well done.

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