Colony hybridization is the method which was first given by the scientists Grinstein and Hogness in the year 1975. This method isolates the specific DNA sequences or genes from the hybrid DNA.
Colony hybridization is carried out by the nitrocellulose filter paper. This technique is very much similar to the replica plating. Colony hybridization involves preparing of replicas on the nitrocellulose membrane filter paper.
In colony hybridization, the bacterial cells are transferred in-situ from the master plate to the nitrocellulose membrane. The nitrocellulose filter paper involves some washing methods, UV exposure and at last hybridization of the desired gene with the specific probe.
The hybridization of DNA is carried out by the radioactive RNA followed by “Autoradiography”. The hybridized DNA is then picked from the reference or master plate for further study.
Content: Colony Hybridization
- Definition of Colony Hybridization
- Transferring medium of Colony Hybridization
- The process of Colony Hybridization
Definition of Colony Hybridization
Colony hybridization is the “Blot analysis technique” where the bacterial cells are transferred from the solid nutrient medium to the absorbent material. Colony hybridization can define as the method for the isolation of the specific DNA sequences or genes from the bacterial cells containing hybrid DNA, by the means of a nitrocellulose membrane filter. The transferring medium then goes through several chemical and physical treatment.
The nitrocellulose filter paper is the transferring medium of the colony hybridization which forms replicas of the master plate. The nitrocellulose acts as a membrane which contains the exact copies of the gene to that of the master plate. Nitrocellulose filter paper acts as the “Blotting pad”.
Ideal properties of the Nitrocellulose filter paper
- The nitrocellulose filter paper is made of 100% pure nitrocellulose, where the cellulose undergoes nitration by the chemical reagent nitric acid.
- This membrane filter provides a high-quality transfer.
- Nitrocellulose filter paper consists of 0.45 µm pore size for efficient transferring.
For colony hybridization, nitrocellulose filter paper goes through several steps like:
- Three times washing of the filter paper with distilled water.
- Placing of filter paper between the absorbent sheets.
- Autoclaving of filter medium at 120 degrees Celsius for 10 minutes.
- Drying of nitrocellulose filter paper in the hot air oven for up to 10 minutes.
The process of Colony Hybridization
The process of colony hybridization involves the following steps:
Preparation of Master plate
First, inoculate the bacterial cell suspension on the solid agar medium to prepare the master plate. After the inoculation, the number of bacterial colonies will develop with different plasmids which refer as “Master or Reference plate”.
Formation of replicas over a nitrocellulose filter
Then transfer the bacterial cells from the master plate on to the membrane or filter by the means of “Nitrocellulose filter”. Press the nitrocellulose filter paper over the surface of the master plate. This compression of the filter membrane will form replicas or copies of the bacterial cells as that of the master plate.
Treatment of filter medium with SDS
After that treat the nitrocellulose filter paper with the detergent-like SDS (Sodium dodecyl sulfate) to lyse the bacterial cells.
Treatment of filter medium with alkali
Treat the filter medium with the alkali like sodium hydroxide in order to separate the DNA into single strands.
Fixation of DNA onto the filter medium
To fix the DNA onto the nitrocellulose filter paper, either bake the filter paper at 80 degrees Celsius or expose it to the UV light.
Addition of radioactive probe
Hybridize the nitrocellulose filter paper containing imprints of the plasmid DNA by the addition of radioactive RNA probe. This radioactive RNA probe will code the desired gene of sequence from the bacterial cells.
Washing and Autoradiography
Wash the filter paper to remove unbound probe particles. After that, expose the nitrocellulose filter paper to the X-ray film by the method refer as “Autoradiography”. The colony which will appear after autoradiography will refer as “Autoradiogram” which carry the genes of interest.
Identification of the desired gene
Then compare the developed autoradiogram with the master plate to identify the colonies containing a gene of interest.
Therefore we can conclude that the colony hybridization method is the “Screening technique” which makes the use of the radioactive probe. The radioactively labelled probe then screen or isolate the particular gene from the number of bacterial colonies.