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Fluorescence in situ hybridization, or FISH, was developed in the early 1980s and has quickly become one of the most widely used techniques in cytogenetics. Labeled probes are used to bind complementary DNA or RNA sequences on a chromosome or in a region within a cell. Earlier, the probes could only be obtained by cloning or reverse transcription of a DNA template. Currently, the probe oligonucleotides can be synthesized synthetically. Additionally, with the advancement of optical techniques,...
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In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled probe. The technique was first used in 1969 for the investigation of nucleic acids. It is currently an essential tool in scientific research and clinical settings, especially for diagnostic purposes.
Types of probes and labels
A probe is a complementary strand of DNA or RNA that binds to corresponding nucleotide sequences in a cell. Many...
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Fluorescence In Situ Hybridization Probe Preparation.

Doron Tolomeo1, Roscoe R Stanyon2, Mariano Rocchi3

  • 1Department of Biology, University of Bari, Via Orabona 4, 70124, Bari, Italy.

Methods in Molecular Biology (Clifton, N.J.)
|December 3, 2016
PubMed
Summary

Genomic clones (GC) are crucial for molecular cytogenetics, especially cancer research. Protocols for DNA extraction, labeling, and hybridization from GCs are summarized for FISH experiments, enabling precise genome mapping and variation analysis.

Keywords:
FISHMolecular cytogeneticsProbe labeling and hybridization

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Area of Science:

  • Molecular Cytogenetics
  • Genomics
  • Biotechnology

Background:

  • The Human Genome Project employed a hierarchical strategy using Bacterial Artificial Chromosome (BAC) and PAC clones for genome sequencing.
  • BAC/PAC clones, with insert sizes from 50 kb to 300 kb, were mapped using Sequence Tagged Sites (STS) to create a physical map.
  • End-sequence information from numerous BACs facilitated precise mapping, even for clones not fully sequenced.

Purpose of the Study:

  • To detail methods for preparing and utilizing genomic clones (GCs) for Fluorescence In Situ Hybridization (FISH) experiments.
  • To highlight the utility of GCs in molecular cytogenetics, particularly for identifying copy number variations and in cancer research.
  • To provide protocols for DNA extraction, labeling, and hybridization from GCs for diagnostic and research applications.

Main Methods:

  • Utilized BAC/PAC clones and fosmid clones (fixed insert size ~40 kb) for high-resolution genome mapping.
  • Sequenced ends of approximately 7 million fosmid clones for precise mapping.
  • Summarized protocols for DNA extraction, labeling, and hybridization of GCs for FISH analysis.

Main Results:

  • A 'golden path' of BACs covering the human genome was established through hierarchical sequencing.
  • End-sequencing of numerous BAC and fosmid clones enabled precise mapping of genomic regions.
  • Genomic clones provide bright FISH signals, proving valuable for molecular and cancer cytogenetics.

Conclusions:

  • Genomic clones are essential tools for high-resolution genome mapping and the detection of copy number variations.
  • The described protocols facilitate the use of GCs in FISH experiments for various cytogenetic applications.
  • GCs are foundational for commercially available labeled probes used in diagnostics and research.