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Localization of Low-Copy DNA Sequences on Mitotic Chromosomes by FISH

  • Miroslava KarafiátováEmail author
  • Jan Bartoš
  • Jaroslav Doležel
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1429)

Abstract

Fluorescence in situ hybridization (FISH) is a widely used method to localize DNA sequences on mitotic and meiotic chromosomes and interphase nuclei. It was developed in early 1980s and since then it has contributed to numerous studies and important discoveries. Over the decades, the protocol was modified for ease of use, allowing for localizing multiple probes simultaneously and increasing its sensitivity and specificity. Despite the continuous improvements, the ability to detect short single-copy sequences of only a few kilobases or less, such as genes, remains limited. Here, we provide a detailed protocol for detection of short, single- or low-copy sequences on plant mitotic metaphase chromosomes.

Key words

Cell cycle synchronization Cytogenetic mapping Fluorochrome Low-copy probe Mitotic metaphase chromosomes Probe purification Post-fixation Quantum yield 

Notes

Acknowledgment

We thank Zdeňka Dubská for technical assistance with cell cycle synchronization. Barley fl-cDNA clones were kindly provided by Kazuhiro Sato (Okayama University, Japan). This work was supported by a grant award LO1204 from the National Program of Sustainability I.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Miroslava Karafiátová
    • 1
    Email author
  • Jan Bartoš
    • 1
  • Jaroslav Doležel
    • 1
  1. 1.Centre of the Region Haná for Biotechnological and Agricultural ResearchInstitute of Experimental BotanyOlomoucCzech Republic

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