High-Affinity LNA-DNA Mixmer Probes for Detection of Chromosome-Specific Polymorphisms of 5S rDNA Repeats in Arabidopsis thaliana

Abstract

Fluorescence in situ hybridization is a standard technique to visualize specific DNA sequences by hybridization with fluorescent probes and, most commonly, relies on DNA probes generated by nick translation. In this chapter, we describe the use of directly labeled LNA-DNA mixmer probes for the rapid detection of repetitive sequences on Arabidopsis thaliana nuclei spreads. We further demonstrate that due to the high thermal stability of the heteroduplexes and the resulting elevated binding affinity of LNA-DNA mixmer probes for their target DNA, these probes can be used to discriminate between repetitive sequences differing by only a few single nucleotide polymorphisms.

Keywords: 5S rDNA; Arabidopsis thaliana; Binding affinity; Fluorescence in situ hybridization; LNA–DNA mixmer probes; Nuclei spreads.