6 -5.3(b).2 Fluorescent in situ hybridization (FISH)

The principle of this method is described in detail in Module 3-Lecture 1. It was first employed with metaphase chromosomes to locate the position of a marker on a chromosome or extended DNA molecule using fluorescent probes leading to low-resolution which was insufficient for the construction of useful chromosome maps. Since 1995, various high resolution techniques have been developed for accurate mapping of chromosomes by altering the chromosomal nature as described below,

1. Mechanical stretching of chromosomes which can be done by altering the isolation method of metaphase chromosomes by centrifugation resulting in the stretching of the chromosomes several times to their normal length. FISH signals are mapped with improved resolution; markers 200-300 kb of length can be mapped by this method.

2.Another approach is to map the non-metaphase chromosomes (those in prophase and interphase), which are sufficiently condensed to be easily identified. Resolution obtained is 25 kb but there is a loss in the characteristic chromosome morphology for which preliminary map information is required to locate the position of markers on a small region of chromosome.

To overcome the above limitation, fiber-FISH has emerged as a high resolution method (>25 kb) which uses DNA prepared by gel stretching or molecular combing and can distinguish markers lying within 10 kb.

Gel stretching:

The chromosomal DNA is suspended in molten agarose and pipetted onto the microscope slide coated with restriction enzyme (inactive stage). With the cooling and solidification of agarose, the DNA molecules become stretched. When magnesium chloride is added, the magnesium ions activate the restriction enzyme which cuts these molecules. With the gradual coiling of molecules, the gaps representing the cuts can be visible under fluorescence microscope (Figure 6-5.3(b).2(i)).

Molecular combing:

In it, silicone-coated cover slip is dipped in DNA solution where these molecules get attached to it by their ends. With the withdrawal of cover slip at a rate of 0.3 mm s^-1, these DNA molecules get retained as an array (comb) of parallel molecules (Figure 6-5.3(b).2(ii)).