Fluorescence in Situ Hybridization

 Instead of a dye, a molecular probe (i.e. a fragment of DNA) binds to the chromosome.

 In early studies, the probe was labeled with a radioactive compound (usually tritium) such that
after 5 to 10 days of exposure to X-ray film, a pattern of silver grains could be detected
identifying the chromosomal location of that probe. (Autoradiography)

 This technique facilitated gene mapping by identifying gene loci (Trask 1991).

 The new technology, fluorescence in situ hybridization (FISH), is quicker, more specific, and
allows the use of multiple probes in a single hybridization procedure.

 In clinical applications, the goal is to determine if a gene or specific mutation is present, so the
molecular probe must be well characterized and specific to the locus in question - most critical
elements in FISH.

 There are three basic types of probes.

1) A unique sequence probe is usually isolated from a cloned disease causing gene (Cherif, 1989;
Linsay, 1993).

2) Chromosome painting probes are actually a cocktail of many unique DNA fragments from along the
entire length of a chromosome such that following hybridization the entire chromosome fluoresces.

3) Repeat Sequence Probes are isolated from telomere or centromere regions.

a) Centromere probes are usually used in chromosome enumeration (i.e. to detect the gain or
loss of specific chromosomes) (Lichter, 1990).

b) Telomere probes are often used to characterize cryptic rearrangements (i.e. to determine if
both telomeres of a chromosome are present and located on the correct chromosome or if a deletion
of rearrangement has occurred.

 Furthermore, instead of radioactively labeled probes, a fluorescent dye is utilized that is

visualized using fluorescence, microscopy (Ledbetter, 1989).

FISH Technique.

 As in the original in situ hybridization, FISH combines the approaches of molecular biology and
made in exactly the same way as a classical cytogenetic study.

 The DNA on the slides is then denatured and a fluorescently labeled single stranded molecular
probe is allowed to hybridize to the chromosomal DNA using an annealing temperature that
favors hybridization of homologous regions of DNA.

 After an appropriate period of hybridization, excess probe is washed away and the
nonhybridized DNA is counterstained with another fluorochrome to allow vizualization of the
entire chromosome complement.

 A fluorescent microscope with light from a 100-w mercury bulb in combination with appropriate
sets of exciter filters allows evaluation of the cells.
  Due to the optical limitations of the glass filters, a maximum of three colors can be visualized on
a typical fluorescent microscope.

 One of the problems with deletion analysis is that it reads lack of signal as positive indication of a
deletion.

 Failure of hybridization may also result in absence of signal.

 To eliminate this as a source of error, a minimum of 20 cells must be evaluated and all cells must
agree in signal count.

 If mosaicism is suspected, additional cells may be surveyed.

 In addition, unique sequence probes are currently used in combination with a control probe.

 The secondary probe is localized to the same arm of the chromosome as the deletion locus.

 It may be labeled with the same fluorochrome as the deletion locus probe, but it has been found
that the interpretation of the hybridization results are easier if the control site probe is labeled
with a different color fluorochrome.

 In all cells evaluated, two clear control signals must be seen, and then the corresponding signals
for the disease locus can be recorded.

 This provides a hybridization control as well as a marker for the chromosome of interest. If a dual
color system is utilized the study may be possible in interphase as well as metaphase cells.

 However, in some cell preparations, very few metaphase cells may be present, sequence or
repeat sequence probes may be used in Interphase nuclei.

 Here, the technical problems are increased due to random loss of signal, extra signals, and
overlapping signals in cells, so additional cells (a total of 50 to 200) must be counted to obtain a
statistically significant result.

Multicolor FISH.

 One of the advantages of FISH over the old in situ hybridization is the ability to hybridize
multiple probes to a single slide and obtain a better understanding of chromosome
rearrangements (Schrock, 1996; Speicher, 1996).

 However, even here there are limits.

 A fluorescent image is the result of the light from the mercury bulb passing through an exciter
filter, hitting the fluorochrome on the slide, and transmitting the appropriate color image to the
eyepiece for viewing.

 Each fluorochrome requires its own filter, so for each additional color desired, the light must
pass through additional thicknesses of glass.

 The optical properties of glass and light therefore limit the total number of viewable colors to
three.
 However, with the advent of computer-assisted systems, multiple colors become possible for a
single hybridization. The computer does not actually “see” more colors but it can detect subtle
differences in shades better than the human eye.

 In a relatively simple example, two fluorochromes are used and mixed in varying proportions to
give a range of colors .

 The computer registers each as a different shade of gray and then assigns a unique color to that
shade for display on the computer monitor.

 A multicolor image can then be printed.

 Combination of fluorochromes have been developed that allows detection of each of the 24
different chromosomes followed by a unique color assignment by the computer (Reid, 1992a,
1992b; divane 1994).

Example:

Multicolor fluorescent in situ hybridization

images of O. officinalis complex