Generation of Densely Labeled Oligonucleotides for the Detection of Small Genomic Elements

Overview

Journal Cell Rep Methods

Specialty Cell Biology

Date 2024 Aug 13

PMID 39137784

Authors

Clemens Steinek

Miguel Guirao-Ortiz

Gabriela Stumberger

Annika J Tolke

David Horl

Thomas Carell

Hartmann Harz

Heinrich Leonhardt

Affiliations

Soon will be listed here.

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.

Citing Articles

Altered enhancer-promoter interaction leads to MNX1 expression in pediatric acute myeloid leukemia with t(7;12)(q36;p13).

Weichenhan D, Riedel A, Sollier E, Toprak U, Hey J, Breuer K Blood Adv. 2024; 8(19):5100-5111.

PMID: 39121370 PMC: 11460460. DOI: 10.1182/bloodadvances.2023012161.

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