In colony hybridization, what detects hybridization?
After transfer and fixation of colony DNA to membrane, hybridization with radiolabeled probe specific for insert is carried out. To visualize successful annealing, membranes are exposed to X-ray film sensitive to beta radiation emitted by isotopes such as 32P incorporated into probe backbone. Regions where probe hybridized produce dark spots on film due to autoradiographic emission, aligning with original colony positions. This X-ray autoradiography provides high sensitivity and spatial resolution. Detection does not rely on ultraviolet fluorescence, colorimetric X-gal cleavage, or direct fluorescent microscopy observations employed for alternative screening modalities.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.