1. Generation of densely labeled oligonucleotides for the detection of small genomic elements.
- Author
-
Steinek C, Guirao-Ortiz M, Stumberger G, Tölke AJ, Hörl D, Carell T, Harz H, and Leonhardt H
- Subjects
- Humans, Genomics methods, Single-Cell Analysis methods, Fluorescent Dyes chemistry, In Situ Hybridization, Fluorescence methods, Oligonucleotides genetics
- Abstract
The genome contains numerous regulatory elements that may undergo complex interactions and contribute to the establishment, maintenance, and change of cellular identity. Three-dimensional genome organization can be explored with fluorescence in situ hybridization (FISH) at the single-cell level, but the detection of small genomic loci remains challenging. Here, we provide a rapid and simple protocol for the generation of bright FISH probes suited for the detection of small genomic elements. We systematically optimized probe design and synthesis, screened polymerases for their ability to incorporate dye-labeled nucleotides, and streamlined purification conditions to yield nanoscopy-compatible oligonucleotides with dyes in variable arrays (NOVA probes). With these probes, we detect genomic loci ranging from genome-wide repetitive regions down to non-repetitive loci below the kilobase scale. In conclusion, we introduce a simple workflow to generate densely labeled oligonucleotide pools that facilitate detection and nanoscopic measurements of small genomic elements in single cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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