A simple non-toxic ethylene carbonate fluorescence in situ hybridization (EC-FISH) for simultaneous detection of repetitive DNA sequences and fluorescent bands in plants

Abstract

The major drawbacks of standard plant fluorescence in situ hybridization (FISH) designed for double-stranded DNA probes include requirement for experimentally determined heat denaturation of chromosomes at high temperatures and at least overnight hybridization. Consequently, processing with chromosomal preparations may easily result in heat-induced deterioration of chromosomal structural details, is time-consuming, and involves the use of toxic formamide and formaldehyde. Here, I have described a simple and appealing non-toxic procedure with ethylene carbonate (EC)—a formamide-substituting solvent and double-stranded repetitive DNA probes. Applying EC as a component of the hybridization solution at 46 °C not only allowed successful overnight hybridization but also gave a possibility to reduce the hybridization time to 3 h, hence converting the technique into a 1-day procedure. Importantly, the EC-FISH tended to preserve well chromosome structural details, e.g., DAPI-positive bands, thus facilitating simultaneous FISH mapping and chromosome banding on the same slide. The procedure requires no formaldehyde and RNA-se treatment of chromosomes, and no heat denaturation of chromosomal DNA. The key condition is to obtain high-quality cytoplasm-free preparations. The method was reproducible in all the plants studied ( Allium , Nigella , Tradescantia , Vicia ), giving a species-specific signal pattern together with clear DAPI bands on chromosomes. The procedure described here is expected to give a positive stimulus for improving gene-mapping approaches in plants. Electronic supplementary material The online version of this article (10.1007/s00709-019-01345-7) contains supplementary material, which is available to authorized users.

Comparison of ethylene carbonate and formamide as components of the hybridization mixture in FISH

“…Cuadrado et al (2009) showed that, regardless of whether hybridization lasted overnight, 2 h, 1 h, or 30 min, they were able to obtain clear hybridization signals. Based on literature data (Matthiesen and Hansen, 2012;Golczyk, 2019) and the results of our work, it seems that the hybridization time may also be reduced in the case of EC-FISH.…”

“…In this study, ND-FISH (non-denaturing fluorescence in situ hybridization) was not tested, because denaturation at 83 °C does not affect chromosome morphology. Golczyk (2019) obtained satisfactory results after hybridization for 3 h. The use of JNK and Bilby probes allowed to obtain positive hybridization results after 90 min of its duration. Thus, it seems that EC-FISH does not only enable avoidance of toxic compounds, but it may also be a time-saving procedure.…”

“…Each of the method modifications obtained specific signals in the centromeric region of all rye chromosomes (Bilby probe) or on 2RL chromosomes (JNK probe), according to the Bilby (Francki, 2001) and JNK (Achrem et al, 2010) sequences localization in Secale vavilovii chromosomes. Golczyk (2019) was the first to present the potential use of ethylene carbonate instead of formamide in a FISH procedure for plant material (Allium, Nigella, Tradescantia, Vicia). The author used probes designed for repetitive sequences (rDNA) and proved that a hybridization mixture with 15 % EC enables omission of the denaturation step.…”

“…However, it should be noted that only repetitive DNA probes were tested and positive results were obtained on the high-quality preparations. Golczyk (2019) analyzed the results of hybridization carried out at two different temperatures (46 °C or 50 °C) in the presence of 15 % EC in the hybridization mixture. The authors also tested two different temperatures (50 °C or 55 °C) of post-hybridization washes in a 2 × SSC solution.…”

Potential of Flow Cytometric Approaches for Rapid Microbial Detection and Characterization in the Food Industry—A Review

“…Hybridization protocols require the use of the toxic solvent formamide for the reliable annealing of oligonucleotide probes to the target site, and studies are being conducted to replace formamide with non-toxic substances and still to achieve reliable annealing of the probes to the target sequence. Matthiesen and Hansen [ 203 ] revealed promising results using ethylene carbonate in non-toxic concentrations instead of formamide for tissue FISH, whereas Kalinka, My, and Achrem [ 204 ] and Golczyk [ 205 ] successfully substituted formamide with ethylene carbonate in plant FISH. Urea was used as a formamide alternative for the detection of gene expression in diverse animal species [ 206 ].…”