This post, is about the cytogenetic technique called FISH, abbreviation for Fluorescence In Situ Hybridization. This technique literally fishes out the location of a particular DNA sequence from the repertoire of the DNA. The bait that is here is labelled stretch of DNA (around 100-1000 bps) called probe and the basic principle which makes this technique work is hybridization or renaturation. That is when a ss DNA fragment finds its complementary sequence on another ss DNA stretch, it will stabilise itself by forming hydrogen bonds with the complementary DNA molecule.
The first FISH experiment was done by Pardue and Gall in year 1969 and since then many modifications have been made.
Let us see what is the process like!!
First of all as mentioned, the bait is the small stretch of DNA of about 100 to 1000 bps called the probe. The probe is either labelled with the fluorescent molecule (florochrome, direct labelling) or is linked with a protein or an antigen molecule ( for indirect labelling). The probe is complementary to the sequence of DNA that we are interested in, hence is responsible for the specificity. The sequence of bases in a probe is of utmost importance and hence probe should be carefully designed. The probe is labelled and hence can be visualized using fluorescent microscope ( fluorescently labelled probes)
In situ hybridization (ISH) techniques involve localization of specific nucleic acid targets within fixed tissues and cells, and no extraction of nucleic material is done. Here the probe is added to the chromosome preparation of the target cell fixed onto a glass (or other) substrate, either in metaphase or even interphase.
As the FISH can be carried out in interphase chromosomes as well, it becomes possible to locate genes in the cells which do not grow rapidly in cell culture or even paraffin embedded tissue samples.
The target DNA and the probes are denatured using heat or chemicals and then mixed together to allow the probe to bind its complementary region. The excess or unbound probes is washed off. The probe will remain bound to its complementary region
As the probe is fluorecently labelled either directly or indirectly (using fluorescently labelled Antibodies or proteins), the binding of the probe to the target DNA can be visualized using a fluorescent microscope.
Fig 2: Steps in FISH.
The number of fluorescent spots present on the chromosomes is analysed. If there is a deletion, only one fluorescent spot will be seen instead of two (in case of normal individual). When there is a duplication, three bright spots will be seen instead of just two (as in the fig 3).
– Advantages of FISH:
- FISH can be carried out using interphase chromosome as well, hence cells need not be cultured or frequently multiply. This also makes it possible to use different tissue samples and sections.
- Labelling probes with different fluorophores allows detection of multiple sequences at a time.
– Applications of FISH:
A. Using Metaphase chromosomes:
1. Localisation of a DNA sequence: The probes when bond to the sequence of DNA on metaphase chromosome, makes it possible to identify the position of the particular DNA sequence in situ on chromosome.
2. Cloned DNA position can be located on metaphase chromosomes
3. Since the exact chromosome and location can be identified, this is useful for mapping of gene
4. Can be used for clinical diagnoses.
B. Using Interphase Chromosomes:
Since there is no need to culture cells, the difficult to culture cells like solid tumor cells, paraffin embedded tissue samples can be processed.
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