To understand how ISH works you will need to understand something about the chemistry of DNA.

The DNA (deoxynucleic acid) in chromosomes is made up of many repeating units of four different "deoxynucleotides", adenine, thymidine, guanine and cytosine, usually abbreviated A, T, G, and C. Deoxynucleotides are complex molecules consisting of a phosphorylated 5-carbon sugar (ribose) with a pyrimidine or purine nucleoside or "base", are linked together by phosphate bonds, forming a "strand" of DNA.

The sequence of these four deoxynucleotides forms the "genetic code" which specifies all your genes and many other characteristics of you DNA. Put simply, the sequence AATGGCATC carries different information than CGGTATACA, even though the nucleotide composition of the two sequences is the same (3As, 2Ts, 2Gs & 2Ts). Each chromosome is made up of two strands of DNA. One DNA strand in a single chromosome may have up to 240,000,000 of these bases, forming a single molecule which is several centimeters long! The two strands are not identical, but "complimentary" and bind to each other in a specific way due to non-covalent bonding affinity between specific bases. For example, the adenine (A) in one strand will bond with a thymidine (T) in the complimentary strand, but will not bond to G or C. The two DNA strands in a chromosome are fully complimentary and a diagram of a small portion of the two strands at very high magnification might look like this: Click to Enlarge Because of base pairing, the length of a DNA strand is usually described in terms of "base pairs" (one base for each strand). The ability of complimentary strands of DNA to recognize each other with very high specificity and to bond tightly together ("hybridize") is a fundamental property of DNA, and is also the basis for "in situ hybridization" (ISH).

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ISH is binding between complementary strands of DNA conducted in the laboratory, where one strand is in cells or tissues and the other is added by the laboratory scientists.

FISH and CISH are varieties of in situ hybridization suitable for use on cell or tissue slides where the read-out is by microscopic examination.

For ISH, a section of chromosomal DNA is selected as the "target" because it is clinically relevant. For instance, if the cytogeneticist wants to know how many copies of chromosome 21 are present in a group of cells, any specific marker segment of DNA from chromosome 21 will be a suitable target. If the cytogeneticist wants to know whether all or a part of the lower section of chromosome 19 (19q) has been lost, they would pick a section of DNA near the tip of 19q. A control target is also selected; in the case of ISH performed to look for trisomy 21, for example, it would be a site on another chromosome.

A relatively short segment of DNA complimentary to the target DNA (i.e. a complimentary DNA sequence), the "probe", is then labeled in one of two ways. For FISH the probe is chemically labeled with a large number of molecules ("fluorophores") which fluoresce with a specific colour when ultraviolet light (invisible to the human eye) is shined on them. Common fluorophores include fluorescein (green) and rhodamine (red). Under the microscope these FISH probes appear as fluorescent colored dots when they are bound to the relevant section of DNA. For CISH the probes are tagged with an enzyme which converts a colorless soluble salt to insoluble, colored crystals; under the microscope the presence of each probe on the microscope slide appears as a small clump of colored crystals.

The actual technique for performing ISH seems relatively simple, but each step must be performed very precisely to obtain correct results. A salt solution containing the labeled probe is layered on top of microscope slides containing the cells or tissues for analysis, and the probe is allowed to bind (hybridize) to the target DNA. The binding step involves heating the slide to dissociate the double-stranded chromosomal DNA and then cooling it gradually to allow the probe to attach by hybridization to its complimentary region on the chromosome. Any residual probe, not bound to the target DNA segment, is then washed off. An example of FISH using one probe to look for an increased number of chromosome 21 and another, as a control, to look for chromosome 13 (which in a viable fetus is always present as a pair) is shown detecting Trisomy 21.