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  Section: General Biotechnology / Genes & Genetic Engineering
 
 
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Techniques of Genetic Engineering

 
     
 

Gene Cloning in Prokaryotes
Success in genetic engineering has been possible due to rapid development in gene cloning methodologies. It is essentially the insertion of a specific fragment of foreign DNA into a cell, through a suitable vector, in such a way that inserted DNA replicates independently and transferred to progenies as a result of cell division. The transformed cells containing DNA after their characterization and confirmation can be used commercially for the production of useful compounds such as insulin, interferon, growth hormones, etc. Maniatis et al. (1976) have described the basic techniques of gene cloning. The principal steps of cDNA preparation shown in Fig. 2.13 are briefly described.

 

Content

Gene cloning in prokaryotes

 

Isolation of DNA to be cloned 

 

Insertion of DNA fragment into vector 

 

 

Use of restriction Linkers

 

 

Use of homopolymer tails

 

Transfer of recombinant DNA into bacterial cells

 

Selection of clones

 

 

Colony hybridization techniques

 

 

In vitro translation technique

 

 

Immunological tests

 

 

Blotting Techniques

 

Recovery of cells

 

Expression of cloned DNA

 

 

Shine-Dalgano sequence

 

 

Expression vectors

Gene cloning in eukaryotes

 

Plant cells

 

 

Yeasts

 

 

Filamentous fungi

 

 

Agrobacterium plasmids

 

 

Plant cell transformation

 

 

Plant cell transformation by ultrasonication

 

 

Liposome mediated gene transfer

 

Animal cell  

 

 

Animal viruses

 

 

Electroporation

 

 

Particle bombardment

 

 

Microinjection

 

 

Direct transformation

Site directed mutagenesis

 

Methods of mutagenesis

 
     
 
 
     



     
 
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