Generation of densely labeled oligonucleotides for the detection of small genomic elements
Cell Rep Methods article from the Leonhardt lab with first author Clemens Steinek
19.08.2024
Clemens Steinek, Miguel Guirao-Ortiz, Gabriela Stumberger, Annika J Tölke, David Hörl, Thomas Carell, Hartmann Harz, Heinrich Leonhardt (2024 Aug 19) Generation of densely labeled oligonucleotides for the detection of small genomic elements. Cell Rep Methods 4(8):100840. DOI: 10.1016/j.crmeth.2024.100840
Summary cited directly from the article:
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.