Analysis of Telomeric G -overhangs by in- Gel Hybridization
| Authors | |
|---|---|
| Year of publication | 2016 |
| Type | Article in Periodical |
| Magazine / Source | Bio-protocol |
| MU Faculty or unit | |
| Citation | |
| Web | http://www.bio-protocol.org/e1775 |
| Doi | http://dx.doi.org/10.21769/BioProtoc.1775 |
| Field | Genetics and molecular biology |
| Keywords | Telomere; G-overhang; In-gel hybridization; Arabidopsis; Telomere analysis |
| Description | 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 variety of organisms ranging from human to plants. |