Genetic Engineering

What is Genetic Engineering?

Genetic engineering also called as recombinant DNA technology, facilitates the manipulation and duplication of DNA pieces, for industrial, medical and research purposes. “Engineering” word in genetic engineering is assigned for designing, construction and manipulation according to a set plan. The benefits of genetic engineering have been realized in wide array of fields.

Genetic engineering allows the study of complex genomes (eukaryotic). Several methods have been developed that allow rapid sequencing of DNA. Genetic engineering has produced a revolution in molecular biology. The techniques of genetic engineering are based on the use of restriction endonucleases from bacteria. These restriction endonucleases are restriction enzymes that recognize specific DNA nucleotide sequences and provide a molecular scissors for cutting DNA at these sites.

Genetic engineering techniques permit the cloning (i.e. the replication of a single molecule) of genes by insertion of the genes into plasmids (via restriction endonucleases) or into bacteriophage lambda. Plasmids are small extra chromosomal circular DNA that is self – replicating. Many of these plasmids carry genes for resistance to antibiotics. The recombinant plasmids carrying eukaryotic genes (desirable genes) are introduced into E.coli and are then selected by use of proper antibiotic.

Colonies can be obtained in which million copies of same molecules are produced. This is called gene cloning. These genetic engineering techniques make possible the use of bacteria to produce proteins of medical importance.

Principle of Genetic Engineering

During sexual reproduction variations are created. Some of the variants are useful and are not produced during asexual reproduction. In hybridization both desirable and undesirable genes multiply in a hybrid. Genetic engineering involves a manipulation of the genetic material (i.e. DNA) towards a desirable end.

In 1972, Stanley Cohen and Herbert Boyer constructed artificial recombinant DNA, for the first time. They got the idea of linking a gene encoding for antibiotic resistance with a native plasmid of Salmonella typhimurium. Plasmid of bacteria is self – replicating circular extra chromosomal DNA bodies. They isolated antibiotic resistance gene by cutting a fragment of DNA from plasmid. This was done with the help of restriction enzymes popularly called as ´Molecular Scissors’. This antibiotic resistant gene fragment was integrated with plasmid DNA. These plasmid DNAs were responsible for transferring the piece of DNA attached to it to host cells, thus acting as Vectors (viz. E.coli cloning vector is pBR322). DNA ligase enzyme helped in joining the DNA fragment with vector. This newly formed circular recombinant DNA was transferred to bacterium E.coli which was able to replicate autonomously in host cell by using DNA polymerase enzyme.

As a result, several copies of this recombinant DNA could be formed. This capability of antibiotic resistance gene in E.coli to form multiple copies is called as cloning of antibiotic resistance gene in E.coli.

Preparation of Genetically Engineered DNA

Following steps are used in preparation of genetically engineered (recombinant DNA).

Step 1: Selection of DNA of interest (commonly called as foreign or target or passenger DNA).

Step 2: A cloning vector or vehicle to carry the inserted pieces of target DNA.

Step 3: Restriction Endonucleases which make internal cuts at specific sites on DNA.

Step 4: DNA ligase, the enzyme that joins the pieces of nucleotides/DNA together.

Step 5: A host to replicate the vector (viz. Plasmid) containing foreign or target DNA. This may be a prokaryotic or Eukaryotic cell.

Step 6: Screening test for recombinants produced by insertion of DNA.

Genetic Engineering

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Process of Genetic Engineering

Process of genetic engineering involves the following steps.

Selection

Selection of DNA fragments coding for proteins of interest are chemically synthesized or isolated from an organism.

Insertion

These desirable DNA fragments are inserted in a restriction endonucleases cleavage site of the vector (plasmid) that does not inactivate any gene required for Vector’s maintenance. DNA ligase joins the pieces of DNA together.

Introduction into host

The recombinant DNA molecules are now introduced into a host (E.coli) to replicate.

Selection and Marking

The recipient host cells that have acquired the recombinant DNA are selected.

Maintaining True Copies

Desired clones are then characterized to ensure that they maintain true copies of the DNA segment that was originally cloned.

The recombinant DNA so produced is introduced into bacterial cells by method called transformation for plasmid DNA & transfection for phage DNA.

Transformation of Plasmid DNA

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