Preparation and Insertional of Recombinant DNA | Obtaining the foreign gene product

Preparation and Insertion of Recombinant DNA into the Host Cell / Organism

• The vector DNA (e.g. plasmid DNA) and alien (foreign) DNA carrying gene of interest are cut by the same restriction endonuclease to produce complementary sticky ends.
• This process of cutting DNA by restriction enzymes is called restriction digestion With the help of DNA ligase enzyme, the complementary sticky ends of the two DNAs are joined (annealing) to produce a recombinant (chimera) DNA (rDNA ).
• The ligase forms new sugar - phosphate bonds to join two DNAs.
• Both direct and indirect methods are used to introduce the ligated DNA into the host cells.
• If a recombinant DNA bearing gene for resistance to antibiotic ampicillin is transferred into E. coli cells, the host cells become transformed into ampicillin resistant cells.
• If such bacteria are transferred on a culture plate containing the antibiotic ampicillin, only the resistant forms marker. will grow and others will die.
• The ampicillin resistance gene in this case is called a selective marker

Obtaining the Foreign Gene Product

• When recombinant DNA is transferred into a bacterial, plant or animal cell, the foreign DNA is multiplied.
• Most of the recombinant technologies are aimed to produce a desirable protein.
• So there is a need for expression of recombinant DNA After the cloning of the gene of interest one has to maintain the optimum conditions to induce the expression of the target protein one should consider producing it on a large scale.
• If any protein encoding gene is expressed in a heterologous host it is known as a " recombinant protein ".
• The cells having cloned genes of interest can be grown on a small scale in the laboratory.
• The cultures may be used for extracting and purifying the desired protein.
• The cells can also be multiplied in a continuous culture system where the used medium is passed out from one side and fresh medium is added from the other side to maintain the cells in their physiologically most active log / exponential phase-- rapid multiplication of the cells.
• This type of culturing method produces a larger biomass to get higher yields of desired protein.

========================================

Mail- indiabiologymanishmevada@gmail.com

Subscribe and Follow For more knowledge of Biology

Join With Me Just Click Hear

Manish Mevada

M.Sc, M.Phil, B.Ed

THANK YOU.