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Related Experiment Videos

High resolution FISH to delineate contiguous and small DNA sequences.

U C Lavania1

  • 1Cytogenetics Division, Central Institute of Medicinal and Aromatic Plants, Lucknow - 226 015, India. lavania@satyam.net.in

Methods in Cell Science : an Official Journal of the Society for in Vitro Biology
|December 13, 2001
PubMed
Summary

Fluorescence in situ hybridization (FISH) using specific DNA probes improved the resolution of weak hybridization sites on rye chromosomes. Pachytene chromosomes offered superior visualization of these sites, aiding in chromosome identification and mapping.

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Area of Science:

  • Cytogenetics
  • Molecular Biology
  • Plant Genetics

Background:

  • Fluorescence in situ hybridization (FISH) is a powerful technique for visualizing DNA sequences on chromosomes.
  • Resolving weak or contiguous hybridization sites can be challenging, especially in condensed somatic chromosomes.
  • Pachytene chromosomes, being more decondensed, offer potential for higher resolution mapping.

Purpose of the Study:

  • To evaluate the resolution of FISH signals for weak and contiguous hybridization sites on rye chromosomes.
  • To compare the effectiveness of different chromosome stages (somatic, meiotic metaphase, pachytene) for FISH analysis.
  • To demonstrate the improved resolution of specific rye repetitive DNA probes on wheat-rye chromosome additions.

Main Methods:

  • DNA: DNA in situ hybridization was performed on somatic, meiotic metaphase, and pachytene chromosomes of rye.

Related Experiment Videos

  • Specific repetitive DNA probes (pSc 200 from the '350 family' and pSc 250 from the '610 family') were used.
  • FISH painting was applied to wheat-rye chromosome additions (5AS/5RL).
  • A wheat RFLP probe (xpr 115) was used for physical localization on barley chromosomes.

    • Main Results:

    • The rye repetitive DNA probe pSc 200 clearly differentiated rye chromosome 5 via two small terminal homologous sites on the long arm.
    • Pachytene chromosomes significantly improved the resolution of these two weak hybridization sites (one distal, one proximal).
    • FISH painting of 5RL in a wheat background at pachytene unequivocally demonstrated higher resolution of these sites.
    • Probe pSc 200 also revealed a large distal telomeric site and a proximal site on 5RS, homologous to pSc 250.
    • Precise experimental conditions allowed spatial resolution of contiguous probes and physical localization of an RFLP probe.

    Conclusions:

    • Pachytene chromosomes provide substantially improved resolution for FISH signals at weak and contiguous hybridization sites.
    • Specific rye repetitive DNA probes can effectively identify and map rye chromosome segments in wheat backgrounds.
    • Optimized FISH protocols enable precise physical mapping of DNA probes, including RFLP markers, across species.