BIOTECH 4

 

COMPETENT HOST

• Since DNA is a hydrophilic molecule, it cannot pass through cell membranes so the bacterial cells must first be made ‘competent’ to take up DNA.
• Several methods are followed to make the bacterial cells competent-
• Treating them with a specific concentration of a divalent cation, such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall.
• Recombinant DNA can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42oC (heat shock), and then putting them back on ice which enables the bacteria to take up the recombinant DNA.
• Recombinant DNA can be directly injected into the nucleus of an animal cell by a method called micro-injection.
• In biolistics or gene gun method, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA
• Disarmed pathogen vectors can be allowed to infect the cell to transfer the recombinant DNA into the host

PROCESS OF RECOMBINANT DNA TECHNOLOGY

Isolation of the Genetic Material (DNA)-

• The cells are broken and opened to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids which can be achieved by treating the cells with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus).
• The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease and purified DNA ultimately precipitates out after the addition of chilled ethanol which can be seen as collection of fine threads in the suspension.

Cutting of DNA at Specific Location-

• Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme, at the optimal conditions for that specific enzyme which results in the fragments of DNA.
• The fragments are separated by a technique known as gel electrophoresis.
• Since DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through agarose.
• The DNA fragments separate according to their size through sieving effect provided by the agarose gel.
• The smaller the fragment size, the farther it moves and the separated DNA fragments can be visualized only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation.
• Bright orange coloured bands of DNA can be observed in an ethidium bromide stained gel exposed to UV light.
• The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece by the process known as