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

 
     
 

Site Directed Mutagenesis
Now, techniques have been developed to mutate the specific portion in the genome in a view to get some novel products of enormous value. Mutations can be done by directing insertions or deletions to a specific site on the DNA but they are unlikely to be of use. These minor changes may have some importance if a single amino acid is altered in a protein in order to improve its properties. For such modifications point-mutations i.e. changing of single nucleotide, is done on specific portion in the gene. Thus, the most suitable method that could bring about point-mutation in a gene is known as oligonucleotide-directed mutagenesis. This technique has potential for protein engineering, the engineered enzymes would be more better than the wild type ones (see Enzyme Technology).

Methods of Mutagenesis
Mutagenesis follows the following steps:

(i)

Synthesis of an oligonucleotide corresponding to mutated nucleotides and its adjacent regions (15-20 nucleotide long).

(ii)

Hybridization of this oligonucleotide with a single stranded clone containing wild type gene at low temperature and high salt concentration. Wild type gene is cloned in Ml3 that is why it is single stranded. The oligomer forms base pairs with its complementary sequence. The oligomer is used as primer for the production of double stranded DNA by DNA polymerase. The double stranded DNA contains two strands. One strand is wild type and the other contains desired point mutations.

(iii)

Transfer of double stranded DNA in E. coli, replication of DNA and production of bacterial colony containing mutant and wild type double stranded DNAs when plated on nutrient medium.

(iv)

Identification of bacterial colonies containing mutated DNA by ‘colony hybridization technique’ at high temperature and low salt concentration.

(v)

Isolation of mutant colony from the master plate and preparation and maintenance of culture for future use.

 

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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