In Situ Hybridization

In situ hybridization was originally described as a generic technique to detect nucleotide sequences (DNA and/or RNA) in tissues or cell preparations. However, over a period of time, especially after the introduction of FISH, the former has almost become synonymous with detection of messenger RNA (mRNA). As elegantly reviewed by McNicol and Farquharson (20), ISH could be applied to a wide variety of biological specimens with diverse visualization systems, including autoradiography, enzymecolorimetry, fluorometry, and chemiluminescence.

From: Molecular Diagnostics: For the Clinical Laboratorian, Second Edition Edited by: W. B. Coleman and G. J. Tsongalis © Humana Press Inc., Totowa, NJ

Table 1 Chromosomal Banding

Giemsa (G) banding: Generically, Giemsa preparation consists of methylene blue, azure B, azure A, azure C and thionin. Essentially they are thiazine molecules with 4, 3, 2, 1, 0 methyl groups, respectively. In addition, there is eosin in the mixture. The staining mechanism involves an interaction between ionically charged thiazins and the phosphate moiety of the DNA sugar phosphate backbone. The staining enhances the banding pattern attained due to the hetero-chromatin and eu-chromatin packaging and the sequence of GC-rich vs AT-rich regions unique to individual chromosomes. This is called G-banding, which is considered to illumninate the AT-rich regions that replicate relatively late in the S-phase during cell cycle.

Reverse (R) banding: A treatment with high-molarity phosphate buffer at high temperature and lower pH and/or staining with acridine orange, produces bands complementary to those revealed by conventional G-banding. This banding pattern termed R-banding primarily demonstrates GC-rich regions that replicate early in the S-phase of the cell cycle. This reverse Giemsa banding method is used to confirm deletions detected by G-banding.

Centromeric (C) banding: The centromeric constitutive heterochro-matin seems to have a cogent binding with nuclear matrix proteins, and as a result, depurinating treatments at low or high pH could enhance the Giemsa staining of the centromeric region, resulting in C-banding, and the rest of the chromosome looking pale.

Quinacrine (Q) banding: The Q-banding employs quinacrine with which the AT-rich region fluoresces brightly, whereas the GC-rich region quenches its fluorescence. Another dye that produces even brighter fluorescence with AT- rich regions is Hoechest 33258. In both cases, a direct interaction with DNA is responsible for banding and no interference with the DNA binding proteins is assumed.

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