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.
Highlights
Fluorescence in situ hybridization (FISH) with doublestranded DNA probes is a powerful and widely employed technique in biological and medical research
In human/animal molecular cytogenetics, ethylene carbonate (EC) is quite frequently used as a component of formamide-free hybridization mixes (e.g., Matthiesen and Hansen 2012; Linhoff et al 2015; Shigeto et al 2016); yet, to my knowledge, there has been no report on application thereof in plants
Other formamidefree FISH protocols with double-stranded DNA probes developed for plants (e.g., Kato et al 2004; Chester et al 2012; Jang and Weiss-Schneeweiss 2015) did not involve EC but included heat denaturation of preparations prior to hybridization with mostly overnight hybridization; the techniques were quite similar to the standard FISH procedure
Summary
Fluorescence in situ hybridization (FISH) with doublestranded DNA probes is a powerful and widely employed technique in biological and medical research. The organic solvent formamide has been widely used as a standard component of hybridization solutions to lower the DNA melting temperature by reducing the thermal stability of the double helix. Formamide lowers the temperature of denaturation and slows down the rate of renaturation, considerably prolonging the time of hybridization. To prevent heatinduced chromosomal damage, preparations are fixed with a formaldehyde solution prior to denaturation, while the time and temperature of denaturation and hybridization are experimentally determined. Processing with chromosomal preparations for standard FISH may still result in heatinduced deterioration/loss of chromosomal structural details; it is time-consuming and involves the use of toxic formamide and formaldehyde, i.e., substances that are dangerous to human health. Formaldehyde vapor is known to cause eye and respiration irritation, dermatitis, asthma, pulmonary edema, and respiratory cancer and to accelerate the speed of leukemia development (Norliana et al 2009; Swenberg et al 2013)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
