Abstract
Telomeric DNA in majority of eukaryotes consists of an array of TG-rich tandem repeats. The TG-rich DNA strand is oriented with its 3’ end towards chromosome termini and is usually longer than its complementary CA-rich strand, thus forming 3’ single stranded overhang (G-overhang). G-overhangs arise from incomplete replication of chromosome termini by the lagging strand mechanism and post-replicative nucleolytic processing. The G-overhang is important for telomere protection as it serves as a binding platform for specific proteins and is required for t-loop formation. Hence, structure of telomeric G-overhang is an important indicator of telomere maintenance and functionality. Here we describe a method for analysis of G-overhangs in a model plant Arabidopsis thaliana by in-gel hybridization technique. This method allows relative quantification of the amount of single stranded telomeric DNA. Short telomeric probes are radioactively labeled and hybridized to DNA under non-denaturing conditions to specifically detect ssDNA. Total telomeric DNA can be measured using denaturing conditions in the same gel and this procedure usually follows the non-denaturing in-gel hybridization. Terminal nature of the ssDNA is verified by exonuclease treatment. This technique was originally developed in yeast and now is used as a major tool for G-overhang analysis in a variety of organisms ranging from human to plants.
Keywords: Telomere, G-overhang, In-gel hybridization, Arabidopsis, Telomere analysis
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Acknowledgments
This protocol was used to analyze G-overhang structure in Arabidopsis (Kazda et al., 2012; Derboven et al., 2014) and was adapted from the previously published studies Dionne et al. (1996), Wei et al. (2002) and Heacock et al. (2007). This work was supported by the EMBO installation grant (1304130933) and the program SoMoPro II (3SGA5833) co-financed by EU and the South Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein.
References
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