Question

How is fluorescence in situ hybridization (FISH) used to produce a spectral karyotype?

Short answer

Answer: FISH is used to produce a spectral karyotype by labeling chromosomes with chromosome-specific probes. These probes are conjugated with fluorescent tags and bind to their target DNA sequences within the chromosomes. This labeling allows for the unique identification and visualization of each chromosome using a fluorescence microscope. The acquired fluorescence images are then analyzed using specialized software, which assigns a pseudo-color to each chromosome and arranges them into a karyotype based on their unique color signature, size, and chromosomal location. This spectral karyotype is useful for detecting chromosomal abnormalities and identifying rearrangements.

Step-by-step solution

Understand the Fluorescence In Situ Hybridization (FISH) technique

Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that is used to detect and locate the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes, which are small pieces of DNA that contain a sequence complementary to the target DNA of interest and are conjugated with a fluorescent tag. When these probes are applied to cells or tissue samples on a slide, they bind to their target DNA sequences, effectively "labeling" the target sequence within the chromosome.

Prepare chromosome-specific probes

In order to generate a spectral karyotype, we must first prepare chromosome-specific probes. These are unique FISH probes that are specific to each individual chromosome in a given species (e.g., humans). Chromosome-specific probes can be derived from entire chromosomes, specific chromosome regions, or unique sequences that are only found on a specific chromosome. These probes are then labeled with different combinatorial mixtures of fluorophores to generate a unique "color" for each chromosome.

Perform the FISH experiment

Now that we have the chromosome-specific probes, we can perform the FISH experiment. First, prepare a slide with fixed cells or tissue sample containing the chromosomes of interest. Next, denature both the DNA in the sample and the probes so that they can hybridize. Apply the probes to the slide, allowing them to bind to their specific target sequences on the chromosomes. Then, wash away any unbound or non-specifically bound probes. Finally, apply a counterstain (e.g., DAPI) to visualize the overall chromosome structure.

Acquire fluorescence images

Using a fluorescence microscope equipped with the appropriate filter sets for each fluorophore used in the labeling of the probes, acquire images of the sample. Typically, images are acquired in a series of channels, each corresponding to a different fluorophore. Using software, these images can be merged into a single, multi-color image, with each chromosome appearing in its unique "color" based on the combination of fluorophores used in the labeling of the probes.

Generate the spectral karyotype

With the fluorescence images, we can now generate the spectral karyotype. This is typically done using specialized software designed for spectral karyotyping analysis. In this process, the software analyzes the unique color signature of each chromosome derived from the combination of fluorophores and assigns a pseudo-color to each chromosome based on that signature. Finally, the software arranges the chromosomes into a karyotype, where the chromosomes are ordered by size and according to their chromosomal location (p and q arms). This spectral karyotype is an effective tool for visualizing chromosomal abnormalities and identifying rearrangements in a sample.