Rapid and Efficient FISH using Pre-Labeled Oligomer Probes

Abstract

Fluorescence in situ hybridization (FISH) is used to visualize the distribution of DNA elements within a genome. Conventional methods for FISH take 1–2 days. Here, we developed a simplified, rapid FISH technique using pre-labeled oligonucleotide probes (PLOPs) and tested the procedure using 18 PLOPs from 45S and 5S rDNA, Arabidopsis-type telomere, and newly-identified Panax ginseng-specific tandem repeats. The 16 developed rDNA PLOPs can be universally applied to plants and animals. The telomere PLOPs can be utilized in most plants with Arabidopsis-type telomeres. The ginseng-specific PLOP can be used to distinguish P. ginseng from related Panax species. Differential labeling of PLOPs allowed us to simultaneously visualize different target loci while reducing the FISH hybridization time from ~16 h to 5 min. PLOP-FISH is efficient, reliable, and rapid, making it ideal for routine analysis, especially of newly sequenced genomes using either universal or specific targets, such as novel tandem repeats identified from whole-genome sequencing data.

New chromosome number and cyto-molecular characterization of the African Baobab (Adansonia digitata L.) - “The Tree of Life”

“…Since there were no visible signs of a satellite distal of the 45S FISH signals for any of the 45S rDNA-bearing chromosomes [Figs. 2 a,e, 3 e (1´, 2´ and 3´), 4f)], we used a separate FISH with the ATRS probe 45 to further investigate the ends of the NORs. As expected, the end of each chromosome arm showed a pair of ATRS signals i.e., one signal on each of the two chromatids (Figs.…”

“…We used 45S rDNA pre-labeled oligonucleotide probes (PLOPs) consisting of four each of 18S and 5.8 rDNA 5′-labeled with AlexaFluor 488, three PLOPs of 5S rDNA 5′-labelled with Cy3 as probes cocktail, and one oligonucleotide probe of Arabidopsis -type telomere repeat sequence [ATRS; (TTTAGGG) n ] 5′-labelled with TexRd-X and Cy3 (Table 2 ; for details see Waminal et al 45 to detect the respective rDNAs and telomeres physical locations on A. digitata chromosomes. The oligonucleotides sequences were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA, USA).…”

“…List of oligonucleotide probes used for fluorescent in situ hybridization* a . * a For details see Waminal et al45. * b For details see Luo et al69.…”

Chromosomal Mapping of Tandem Repeats Revealed Massive Chromosomal Rearrangements and Insights Into Senna tora Dysploidy

“…PLOPs for StoTR01_86, StoTR03_178, StoTR04_55, StoTR05_180, StoTR06_159, and StoIGS_463 were designed using the CLC Main Workbench and were synthesized by Bioneer (Daejeon, South Korea). PLOPs previously developed for the coding regions of the 5S rDNA and 45S rDNA, and the Arabidopsis -type telomeric repeat, which has the same sequence as StoTR02_7_tel, were used to localize these repeats (Waminal et al, 2018a ). Details of the designed PLOPs are summarized in Supplementary Table 1 .…”

“…A rapid FISH method was performed for PLOP-labeled probes (Waminal et al, 2018a ). For PCR amplicon probes labeled via nick-translation, FISH was performed according to a modified procedure of Waminal et al ( 2020 ).…”