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.
» 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