Fluorescent In Situ Hybridization (FISH)

FISH is a technique that is used to detect the presence of specific DNA sequences on chromosomes out of large number of fragments of similar size. It uses fluorescent probes that bind to those parts of chromosome which have similar base sequences. The detection of base sequences on a combed DNA molecule is done by first hybridizing the known base sequences (the probes) with the combed DNA (matrixDNA or target DNA).

The method comprises of three basic steps: fixation of a specimen on a microscope slide, hybridization of labelled probe to homologous fragments of genomic DNA, and enzymatic detection fluorescent detection of the tagged target hybrids. Normal hybridization requires the isolation of DNA or RNA, separating it on a gel, blotting it onto nitrocellulose and probing it with a complementary sequence.

The most common tissue sections used with in situ hybridization are:

a) Frozen sections. Fresh tissue is frozen rapidly and then embedded in a special support medium for cryosectioning. The sections are rapidly fixed in 4% paraformaldehyde prior to hybridization.

b) Paraffin embedded sections. Sections are fixed in formalin and then embedded in wax paraffin before being cut into sections.

c) Cells in suspension. Cells can be cytospun onto glass slides and fixed with methanol.

PROBES

Probes are fragments of cloned DNA that are isolated, purified, and amplified for use in the hybridization. Probes are complimentary sequences of nucleotide bases to the specific mRNA sequence of interest. These probes can be as small as 20-40 base pairs or be up to 1000 bp. These fragments should not be so large as to impede the hybridization process. Probes of different types can be used to detect distinct DNA types.

There are essentially four types of probes that can be used in performing in situ hybridization:

1. Oligonucleotide probes are produced synthetically by readily available deoxynucleotides but the specific nucleotide sequence you wish to prepare should be known. These probes have the advantages of being stable, resistant to RNases and are small, generally around 40-50 base-pairs and hence allow easy penetration into the cells or tissue of interest. They are available for purchase and faster and less expensive to use. Unlike RNA probes, oligonucleotide probes can be designed to selectively recognize members of closely related gene families.
2. Single stranded DNA probes. These are much larger, probably in the 200-500 bp range. They can be produced by reverse transcription of RNA or by amplified primer extension of a PCR-generated fragment in the presence of a single antisense primer. They require time to prepare, need expensive reagents and a good molecular skills.
3. Double stranded DNA probes. These can be produced by the inclusion of the sequences of interest into bacterial cell, which is replicated, lysed and the DNA extracted, purified and the sequence of interest is excised with restriction enzymes. On the other hand, if the sequence is known then by designing appropriate primers one can produce the relevant sequence very rapidly by PCR. Because the probe is double stranded, denaturation or melting has to be carried out prior to hybridization.
4. RNA probes (cRNA probes or riboprobes). RNA probes have the advantage that RNA-RNA hybrids are very thermostable and are resistant to digestion by RNAses. This allows the possibility of post-hybridization digestion with RNAses to remove non-hybridized RNA and therefore reduces the possibility of background staining. These probes however can be very difficult to work with as they are very sensitive to RNases (ubiquitous RNA degrading enzymes).

LABELLING

To see where the probe has hybridized within the tissue section or within cells, you must attach to the probe with an easily detectable substance or “label” before hybridization. Classically oligonucleotide probes have been either 5′ or 3′ end-labelled or 3′ tailed with modified nucleotides that have a “label” attached that can be detected after the probe has hybridized to its target.

Traditionally oligonucleotide probes have been radiolabelled, for instance 35Sulphur (35S) is the most commonly used radioisotope because its high activity is necessary to detect transcripts present in low amounts. Both radiolabels and non-radioactive labels are “attached” to the single stranded oligonucleotide probe by using the enzyme terminal transferase to add a tail of labelled dioxy nucleotides to the 3′ end of the oligonucleotide. Radiolabelled probes are visualized by exposure of the tissue section or cells against photographic film which is then developed.

In contrast to adding the labeled nucleotides to either end of the oligonucleotide probe it is also possible to have labels incorporated into the oligonucleotide when it is being synthesized, for example by adding biotin- or FITC-labeled dATP in place of non-labeled dATP during synthesis so that a label or “tag” appears every time that the ATP nucleotide appears in the probe sequence.

HYBRIDIZATION

Hybridization involves mixing the single strand probes with the denatured target DNA. Denaturation of the DNA is obtained by heating, which separates the two strands, and allows access of the single strand probes to their respective complementary combed single strand.

The factors that influence the hybridization of the oligonucleotide probes to the target mRNAs are: Temperature, pH, monovalent cation concentration and presence of organic solvents.

The following is a typical hybridization solution at temperature of 37oC and an overnight incubation period.

Dextran sulphate. This is added because it becomes strongly hydrated and therefore effectively increases the probe concentration in solution resulting in higher hybridization rates.

Formamide and DTT (dithiothreitol). These are organic solvents which reduce the thermal stability of the bonds allowing hybridization to be carried out at a lower temperature.

SSC (NaCl + Sodium citrate). Monovalent cations interact mainly with the phosphate groups of the nucleic acids decreasing the electrostatic interactions between the two strands.

EDTA. This is a chelator and removes free divalent cations from the hybridization solution, because they strongly stabilize duplex DNA.

 Following hybridization the material is washed to remove unbound probe or probe which has loosely bound to imperfectly matched sequences.

OBSERVATION

Observation of the hybridized sequences is done using epifluorescence microscopy. White light from a source lamp is filtered so that only the relevant wavelengths for excitation of the fluorescent molecules reach the sample. The light emitted by fluorochromes is generally of larger wavelengths, which allows the distinction between excitation and emission light by means of a second optical filter. Therefore, it is possible to see bright coloured signals on a dark background. It is also possible to distinguish between several excitation and emission bands, thus between several fluorochromes, which allows the observation of many different probes on the same target.

As mentioned, radiolabeled probes are detectable using either photographic film or photographic emulsion.

The fluorescent labels described above are detectable by using a fluorescent microscope to examine the tissue on which the labeled oligonucleotide probe has hybridized. The use of fluorescent labels with in situ hybridization has come to be known as FISH(fluorescent in situ hybridization) and one advantage of these fluorescent labels is that two or more different probes can be visualized at one time.

In contrast both Biotin and DIG labeled oligonucleotide probes generally require an intermediate before detection of the probe can occur and they are thus detected indirectly like a typical immunocytochemistry protocol. Biotin is the common compound used in the labelling of oligonucleotide probes. Linked to ATP it can be detected with antibodies but more often with a glygoprotein Avidin from egg white or Streptavidin from the fungi Streptomyces avidinii, as they have a high binding capacity.