Some Peculiarities in Application of Denaturating and Non-Denaturating In Situ Hybridization on Chromosomes of Cereals

Abstract

Non-denaturing fluorescent in situ hybridization (ND FISH) is a convenient method of cytogenetic research. Compared to the standard method, ND FISH is fast and easy to perform and requires less time, reagents, and tools. Thus, it is gaining increasing popularity among different groups of scientists and is used to accomplish various scientific tasks. However, when using this method to visualize the chromosomes of wheat and its wild relatives, we faced some peculiarities of its application when oligonucleotide probes are used. In this paper, we compare the three following methods: two different versions of denaturating and non-denaturating FISH. In the standard procedure and its modifications, chromosomes are treated with formamide at high temperature that results in the denaturation of supercoiled DNA of plant chromosomes. In the non-denaturing FISH, this step is omitted, which makes it possible to keep the native chromosome structure and, thus, is more time and cost effective. In our work, all methods demonstrated their efficiency. Non-denaturing FISH is characterized by ease and convenience but less reproducibility in a series of experiments. The standard protocol and its modifications are most stable and reliable, but negatively affect chromosome morphology. In successive hybridizations on the same slide (sequential FISH), we recommend a combination of these methods, with primary testing using a standard protocol and subsequent hybridization using the ND-FISH method.