Biotechnology and Bioinformatics (1+1)

2.1.5.1 Colony hybridization

It is the preferred choice for screening the colonies to identify and isolate the colony which contains the desired gene.

• In this technique the cells are first plated on selective plates.
• A replica of the colonies is made on nitrocellulose filter disc which is placed on the surface of a second plate.
• The colonies are allowed to grow on the master plate and the nitrocellulose disc.
• The disc is then removed and placed in alkali to lyse the bacteria in situ and to denature their DNA.
• The single-stranded DNA binds to the nitrocellulose filter in the position originally occupied by the bacterial colony.
• The filter is than baked at 80⁰C, following which it is incubated with a solution containing the radiolabelled c DNA probe under conditions which favour nucleic acid hybridization.
• The unhybridized material is removed by extensive washing, thus allowing the identification of colonies containing sequence s complementary to the probe by autoradiography .
• Colonies which give a positive autoradiograph signal can then be picked from the master plate and cultured in order to provide sufficient cells carrying the desired gene.