Applications of Blotting and Hybridization Techniques
1. Southern blotting technique is widely used to find specific nucleic acid sequence present in different plant species.
2. Northern blotting technique is widely used to find gene expression and regulation of specific genes.
3. By using blotting technique we can identify infectious agents present in the sample.
4. We can identify inherited disease. 5. It can be applied to mapping restriction sites in single copy gene.
Disadvantages of Blotting and Hybridization Techniques
1. The process is a complex, cumbersome and time consuming one.
2. It requires electrophoretic separation.
3. Only one gene or RNA can be analysed at a time.
4. Gives information about presence of DNA, RNA or proteins but does not give information about regulation and gene interaction.
Dot Blotting Techniques – The drawbacks of blotting techniques have lead to the development of dot blotting technique which is more advanced, less time consuming, accurate and applicable to a wide variety of gene/source simultaneously. The dot or slot blotting technique is the most widely used of all techniques for analysing. None of the blot methods require electrophoresis prior to blotting and hybridization. Hybridization of cloned DNA without electrophoretic separation is called as dot blotting.
Plaque or Colony Blotting Techniques – This method was first developed by Granstiens and Hogness (1975). This method is used to identify which colony of bacteria contains the DNA of interest among thousands. In this procedure, the bacterial colonies to be screened are transferred onto nitrocellulose or nylon membrane by using replica plating.
Due to the negative charge of the cell surface, some cells bind to the nitrocellulose membrane. Then the membrane is placed in a solution of 0.5 N NaOH to break the cell surface, convert
dsDNA to ssDNA and to bind DNA to the membrane. Later, the membrane is transferred to a solution containing protease solution after neutralizing with neutralization solution.
The DNA is fixed tightly to membrane by either W cross linking or oven baking. This membrane is used for hybridization with a probe and analysed by using autoradiography or biotin method for positive hybridization. A colony whose DNA print (as replica plating provides a replica print master plate colony on the membrane) gives a positive hybridization can be picked from the master plate.
Plaque blotting is similar to colony blotting; the only difference is that instead of bacterial colony, a plaque is transferred onto the membrane. Benton and Davis developed this method in 1977. The greatest advantage of this method is that several identical DNA prints can be easily made from a single master plate containing bacteria/plaques which are to be made.
Dot Plot Assay Techniques – This method is widely used to hybridize DNA from a single cell type against a wide variety of probes, for example, for a viral infection which cannot be identified by normal conventional methods or if we want to know what all genes are expressed in a single cell type (e.g. brain cell).
Cell type or cells that are to be screened are placed on the membrane as ‘dot’ in the order of rows and columns. Then the cells are denatured by using enzymes or detergents (SDS) and DNA is fixed by using W – cross link or oven baking. This membrane is then used for hybridization by using probes (which are specific to a gene).