Biology Biotechnology Principles class 12 Part 5

Selection of recombinant formed can be done by one of the following methods:-

a)Inactivation of antibiotics

• If foreign DNA lights at the BamHl site of tetracycline resistance gene in the vector pBR322, The recombinant plasmid loses the tetracycline resistance due insertion of foregine DNA.
• It can still be selected out from non-recombinant one by plating the transmission on ampicillin containing tetracycline.
• The transformation growing on ampicillin medium are then transferred on to a medium containing tetracycline.
• The recombination can grow in a ampicillin containing medium but not on the containing tetracycline where non- recombinants can grow on the medium containing both the antibiotics and thus recombinant are selected.
  b)Insertional inactivation
• On the basis of colour production in the presence of chromogenic substrate the recombinants and non recombinants can also be differentiated.
• here , a recombinant DNA is inserted within the coding sequence B- galactosidase which results into inactivation of the enzymes.

Competent Host ( For transformation with Recombinant DNA) –

DNA being a hydrophilic molecule . cannot pass through cell membranes.
Therefore the bacteria should be made competent to accept DNA molecules.
Competency is the ability of a cell to take up foreign DNA.

The cell is made competent by the following method:
a)Chemical method
b)Physical method

Chemical method –

• The cell is treated with specific concentration of a divalent cation such as calcium to increase pore size in cell wall.
• The cel are incubated with recombinant DNA on ice followed by placing them briefly at 42 degree celsius and then putting its back on ice. This is called heat shock treatment.
• The bacteria now take up the recombinant DNA.

Physical methods –

• The physical method include
• Micro-injection method : – Recombinant DNA is directly injected into the nucleus of an animal cell.
• Biolistic gene gun method :- cell are bombarded with high velocity micro particle of gold or tungsten coted with DNA in plant.
• Note: Disarmed pathogen vector are also used to transfer y DNA.