Analysis of Telomeric G -overhangs by in- Gel Hybridization

VALUCHOVÁ, Soňa, Elisa DERBOVEN and Karel ŘÍHA. Analysis of Telomeric G -overhangs by in- Gel Hybridization. Bio-protocol. Sunnyvale: Bio-protocol LLC, 2016, vol. 6, No 7, p. nestránkováno, 9 pp. ISSN 2331-8325. Available from: https://dx.doi.org/10.21769/BioProtoc.1775.

Basic information

• Original name: Analysis of Telomeric G -overhangs by in- Gel Hybridization
• Authors: VALUCHOVÁ, Soňa (703 Slovakia, belonging to the institution), Elisa DERBOVEN (40 Austria) and Karel ŘÍHA (203 Czech Republic, guarantor, belonging to the institution).
• Edition: Bio-protocol, Sunnyvale, Bio-protocol LLC, 2016, 2331-8325.

Other information

• Original language: English
• Type of outcome: Article in a journal
• Field of Study: Genetics and molecular biology
• Country of publisher: United States of America
• Confidentiality degree: is not subject to a state or trade secret
• WWW: URL
• RIV identification code: RIV/00216224:14740/16:00093962
• Organization unit: Central European Institute of Technology
• Doi: http://dx.doi.org/10.21769/BioProtoc.1775
• Keywords in English: Telomere; G-overhang; In-gel hybridization; Arabidopsis; Telomere analysis
• Tags: rivok

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 variety of organisms ranging from human to plants.