Fish Techniques

The detailed protocols and their applications of the FISH techniques have been described in a recently published book entitled ''Molecular Cytogenetics: Protocols and Applications.''1-1-1

FISH with Unique DNA Sequences

FISH with unique DNA sequences represents the most basic technique. The DNA segment used as a probe may represent a functional gene or a particular chromosome region or locus. The basic steps of FISH procedure include labeling of DNA probes, preparing interphase or metaphase chromosome slides, in situ hybridization, and visualization with a fluorescent microscope.[2]

Chromosome Painting

Chromosome painting refers to a FISH procedure using probes generated from specific chromosome libraries, Alu and L1 PCR or chromosome microdissection.1-3-1 When the probe contains unique and repetitive sequences from an entire chromosome, whole chromosomes of a homologous pair in metaphases are illuminated with fluorescence (painted). The short or long arm of a particular chromosome can be painted with an arm-specific probe. Using a device containing a 3 x 8 array, all 24 chromosomes can be painted simultaneously and detected sequentially on a single slide using a standard fluorescent microscope.

Spectral Karyotyping

Spectral karyotyping (SKY) is an automated chromosome painting procedure using a probe mixture containing representative DNA for each of the 24 human chromo-somes.[4] The probes are directly labeled with the combinations of five fluorochromes. Through computer classification of the spectra, all 24 human chromosomes can be simultaneously visualized in different colors. SKY has been proven to be a powerful tool for the characterization of complex chromosomal rearrangements in cancer cells and de novo constitutional structural abnormalities. The sensitivity of SKY to visualize DNA alterations is about 1-2 Mb in size.[5]

Multiple Color FISH

Multiple color FISH (M-FISH) is also called multifluor FISH and multiplex FISH.[6] Similar to SKY, the probes are labeled with the combinations of multiple fluoro-chromes. In contrast to spectral analysis, 24 chromosomes in unique colors are detected by a series of fluorochrome-specific filters with the assistance of computer software.

FISH Following Microdissection (MicroFISH)

To perform MicroFISH, a whole chromosome, a marker, or a particular chromosome band is scraped from metaphase spreads using a micromanipulator.1-7-1 The scraped DNA is amplified by polymerase chain reaction (PCR) and labeled as probes. FISH of such probes with normal reference metaphase chromosomes reveals the composition of chromosomes or the chromosome regions in question. This technique is useful for characterizing structural rearrangements and marker chromosomes. It is also a tool to produce specific painting probes for individual chromosomes, chromosomal arms, or regions.

Comparative Genomic Hybridization

Comparative genomic hybridization (CGH) compares test DNA with normal reference DNA.[8] The test DNA is traditionally labeled with a green color and the reference DNA with a red color. The DNA mixture is then hybridized to normal metaphase chromosomes prepared from a blood culture. By measuring the ratio of green to red color, gains or losses of chromosomes or chromosomal regions in the test DNA can be detected. The size of DNA segments that CGH can detect was estimated to be in the range of 10-20 Mb. CGH has been widely used in the investigations of solid tumors. CGH is also useful in the characterization of de novo unbalanced structural abnormalities.

Primed In Situ Labeling

Primed in situ labeling (PRINS) refers to a process of reannealing the short oligonucleotide primers to target sequences in situ, and followed by elongation of the sequences with a Taq polymerase and simultaneous labeling of the target sequences with a fluorochrome.[9] PRINS primarily targets short stretches of a-satellite DNA unique to each chromosome. This technique has been used as an efficient alternative tool to detect aneuploidies.

Fiber FISH

This is a procedure of hybridization of DNA probes to extended chromatin fibers (i.e., free chromatin released from lysed cells) on a microscope slide.[10] A modified method is to hybridize probes to unfixed DNA fibers prepared from cells embedded in pulsed-field gel electrophoresis (PFGE) blocks. Fiber FISH has been used for high-resolution gene mapping, and for direct visualization of gene duplication and chromosome breakpoints involved in translocations.

Color Banding

This technique is also called cross-species FISH (Rx FISH) because it utilizes DNA obtained from flow-sorted gibbon chromosomes by PCR amplification.1-11-1 The genome of gibbons has a high degree of homology with human DNA but with extensively rearranged chromosomes. When hybridized with a set of gibbon DNA probes labeled with a combination of FITC, Cy3, and Cy5, human metaphase chromosomes show a distinctive color banding pattern. A high-resolution multicolor banding can also be achieved by using the microdissection-based probes.[12]

Multitelomere FISH

The technique of multitelomere FISH utilizes a device containing 41 subtelomeric probes for all 24 different chromosomes (not including the short arms of acrocen-trics).[13] The probes for the short arms and the long arms are dual-labeled with a green and a red fluorochrome, respectively. This allows the detection of submicroscopic deletions or translocations in all subtelomeric regions with a single hybridization.

Simultaneous Immunophenotyping and FISH

This is a simultaneous analysis of cell surface immuno-logic markers and interphase FISH. The strategy of combining immunophenotyping with FISH enables correlation of chromosome aberrations of interest with cell lineage and differentiation stages of tumor cells, and therefore provides a useful tool for studies of leukemia and lymphomas.[14]

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook

Post a comment