Comparative Genomic Hybridization A

genomewide screening of the gains or losses of the specific chromosomal regions came into sight with the development of comparative genomic hybridization (CGH). (for review, see refs. 38 and 47). Importantly, such screening is possible from interphase cells, where CGH is superior over M-FISH, SKY, or

Table 4

Common Skepticism Regarding FISH

Q 1. How stable are the probes? Can I freeze-thaw them? Can I store them diluted?

A: Generally recommended storage temperature is -20°C, at which the probes are stable. They can be repeatedly frozen and thawed. Undiluted storage conditions are recommended for better preservation of the FISH probes

Q 2. How long can I store my specimen slides and still hybridize them effectively?

A: The slides stored at -20°C would hybridize with FISH probes, even after a period well over 6 mo.

Q 3. Is sequential hybridization possible on the same specimen? Can FISH be combined with G-banding?

A: Sequential hybridization with different probes is possible as well combination with G-banding possible. For technical details, see ref. 42.

Q 4. How long can the FISH signals be preserved following initial hybridization reaction?

A: By storing slides at -20°C and minimizing the exposure to high-intensity ultraviolet light of the mercury lamp, the FISH signals could be preserved for months. Photobleaching is known to occur gradually over time and with frequent light exposure.

Q 5. Do the CEP and WCP probes crossreact to other human chromosomes?

A: Some crossreactivity with other human chromosomes may occur with the WCP probes. The crossreactivity could be reduced by using human Cot-1 DNA in the probe hybridization mixture.

COBRA-FISH. The technique of CGH relies on a competitive hybridization of test DNA and reference normal DNA that are differentially labeled with two fluorophores (typically Cy-3 with green fluorescence and Cy-5 with red fluorescence, respectively) and then hybridized concurrently in a 1 : 1 proportion to a target normal metaphase chromosome preparation. The computer-assisted quantification of the relative binding ratio of the test vs control over the entire length of each chromosome determines the loss or gain in a particular region in the test DNA. A major drawback of CGH is that if the size of the cytogenetically altered clone in a test specimen is small, it could easily be missed (Table 5). Therefore, in the case of solid tumors, to avoid normal cell contamination, a microdissection of a histopathologically well-defined tumor area is recommended. A plethora of reports are available on CGH analysis in solid tumors and hematological malignancies as reviewed by Zitzelsberger et al. (47), It is noted that a confirmatory test using FISH or PCR- based techniques is highly desirable to ascertain the abnormalities pointed by CGH.

A recent advance in CGH is seen in the development of CGH-based microarrays that use definitive DNA clones immobilized on a solid phase as the hybridization targets (50,51). Although circumventing the reliance on metaphase and better defining the hybridization this technique entertains the possibility of high-throughput analysis. The recent genomic array contains approx 2400 target BAC clones to assess the DNA copy number across the human genome and has the ability to detect single-gene copy gains and losses in a homogeneous DNA specimen (51).

5.3. FIBER-FISH The term "fiber-FISH" is used to describe high-resolution FISH-based genomic mapping of a less condensed, deproteinated, stretched chromatin fiber that is immobilized on a solid phase. A serial treatment of interphase cells on a glass slide with a cell lysis agent, deproteination in a high salt solution, followed by ultraviolet (UV) irradiation and air-drying gives long DNA fibers immobilized on the glass surface. Using FISH probes to hybridize with such DNA fibers, it is hoped that a fluorescent color bar code could be generated of about 400 kb in size that then could be used to screen the clinical DNA sample for gene rearrangements. The resolution of rearrangements of a few kilobases is thus expected to be achieved by fiber-FISH. (52,53).

6. ISH/FISH SIGNAL AMPLIFICATION

In situ hybridization for low-abundance mRNA species as well as interphase cytogenetics signals are often very small and might need additional signal amplification for a meaningful visualization. There are two broad approaches for accomplishing ISH/FISH signal amplification (for a detailed review, see ref. 54). In the first approach of 'target sequence in situ amplification,' the sequence of interest could itself be amplified in situ using a specific primer and labeled nucleotides, whereas in the other approach of posthybridization signal amplification, a single hybridization reaction is used with a reporter system that causes enhanced deposition of fluorochrome.

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