In this article we will discuss about the process of in situ hybridization in nucleic acids.
The mammalian and other higher eukaryons system contains larger DNA molecule. Some times larger than 50,000 kb. Hence, non-conventional methods like gel electrophoresis is not feasible.
The gene location on these larger molecules can be identified by in situ hybridization which can prove accurate information regarding the position of cloned gene. In situ hybridization involved standard light microscopy used to observe chromosome in the cells that are in the process of division.
This technique enables us to visualise directly the location of a cloned gene in a light microscopic image of the chromosome. In the process of in situ hybridization, cells are first treated with fixatives and attached to a glass slide and then incubated with sodium hydroxide and ribonuclease to denature DNA and degrade RNA. Due to alkaline treatment base pairing between the individual polynucleotide strands is broken.
A complementary strand of cloned gene is labelled and mixed with chromosome preparation on the slide. When probes make a contact with denatured DNA within the chromosomes, hybridization takes place between cloned gene and chromosomal copy.
A dark spot on the X-ray film appeared when subjected for autoradiography. The position of dark spot indicated the location of a cloned gene in the chromosome (Fig. 13.11).
In situ hybridization with radioisotope labelling is comparatively different. It is very useful in locating the position of number of genes. An alternative radioactive probe is fluorescent marker. It can be attached to the probe and mixed with chromosomal DNA to facilitate hybridization process and it can be observed directly by designed microscope.
