Rapid and Efficient FISH using Pre-Labeled Oligomer Probes

Nomar Espinosa Waminal,Nam-Soo Kim,Remnyl Joyce Pellerin,Tae-Jin Yang,Jee Young Park,Hyun Hee Kim,Murukarthick Jayakodi

doi:10.1038/s41598-018-26667-z

Nomar Espinosa Waminal, Nam-Soo Kim + Show 5 more

Open Access

https://doi.org/10.1038/s41598-018-26667-z

Copy DOI

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.

Highlights

Fluorescence in situ hybridization (FISH) is a powerful technique for visualizing the chromosomal location of a target DNA sequence in its native cellular environment
We demonstrate a variety of applications for pre-labeled oligonucleotide probes (PLOPs)-FISH for examining universal targets, as well as species-specific high copy number tandem-repeat blocks that were newly identified from next-generation sequencing (NGS) platform-WGS data
The reference mapping generated a 1,851-bp, 201-bp, and 124-bp consensus sequences for 18S, 5.8S and 5S ribosomal DNAs (rDNA), respectively, with reads derived from fungi, animals, and plants (Fig. 1, Supplementary Fig. S1, and Supplementary Table S2)

Summary

Introduction

Fluorescence in situ hybridization (FISH) is a powerful technique for visualizing the chromosomal location of a target DNA sequence in its native cellular environment. In nick-translation, probes are labeled with nucleotide analogs conjugated with either a hapten or a fluorochrome, which is referred to as indirect and direct labeling, respectively[11,13]. The use of telomeric sequences has been vital for elucidating the evolution of some plants as well as the pathophysiology of certain diseases such as cancer[21,22] These sequences, such as TTAGGGn and TTTAGGGn, which are found in many vertebrates and plants, respectively, are vital for chromosome integrity and are generally present in chromosome termini, recent studies have revealed several variations from these canonical repeat sequences[21]

Methods

Results

Conclusion