#FISH

Transcriptome analysis

M-FISH is most suitable for:

M-FISH generates a comprehensive spectral karyotype where each chromosome pair exhibits a distinct combinatorial fluorescent code. This permits detection of numerical aneuploidies, unbalanced translocations, marker chromosomes, and cryptic rearrangements that underlie inherited syndromes such as Down, Turner, Klinefelter, and microdeletion disorders. Because it interrogates entire genomes at chromosomal resolution rather than single nucleotide changes or protein interactions, it excels for constitutional cytogenetic screening. SNP identification requires sequencing or arrays, while transcript length measurement and protein interaction studies employ entirely different transcriptomic and proteomic platforms.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Transcriptome analysis

What kind of microscope is used in FISH?

FISH signals are inherently fluorescent, generated by fluorophores such as FITC, TRITC, Texas Red, Cy3, and Cy5 attached to probes or detected via fluorescent antibody sandwiches. These faint nuclear signals require excitation with specific wavelengths and emission filter sets, which only fluorescence microscopy provides. Confocal microscopy further improves signal-to-noise ratio and optical sectioning, crucial for interphase nuclei and three-dimensional tissue FISH. Bright-field, electron, and phase-contrast microscopes cannot excite or separate fluorochrome emission, thus lacking capability to resolve locus-specific fluorescent spots against dark background.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Transcriptome analysis

Which of the following is INCORRECT about FISH?

Fluorescence In Situ Hybridization is based on nucleic acid hybridization principle. A denatured target chromosomal DNA is annealed with complementary DNA or RNA probes directly conjugated or hapten-labeled for fluorochrome detection via avidin or secondary reagents. Visualization requires fluorescence microscopy, and stringent post-hybridization washes remove non-specifically bound probes to reduce background. Fluorescence-tagged antibodies are characteristic of immunofluorescence or immunohistochemistry for proteins, not essential for standard FISH chemistry. Although indirect FISH may use antibody-mediated detection layers, antibodies are not an obligate component of the core technique.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Transcriptome analysis

Which fluorescent method is used to detect chromosome abnormalities using DNA probes?

Fluorescence In Situ Hybridization combines cytogenetics with fluorescence detection. Denatured metaphase chromosomes or interphase nuclei are hybridized with fluorescently labeled locus-specific, centromeric, or whole-chromosome painting probes. Upon specific binding, signals are visualized by fluorescence microscopy, allowing detection of aneuploidies, deletions, duplications, translocations, and amplifications. Unlike SAGE or CGH, FISH preserves cellular morphology and provides single-cell resolution. Comparative genomic hybridization assesses copy number changes but not balanced rearrangements with the same morphological precision afforded by probe-based in situ labeling and visualization.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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