Analysis of Telomeric G -overhangs by in- Gel Hybridization

J 2016

Analysis of Telomeric G -overhangs by in- Gel Hybridization

VALUCHOVÁ, Soňa; Elisa DERBOVEN a Karel ŘÍHA

Základní údaje

Originální název

Analysis of Telomeric G -overhangs by in- Gel Hybridization

Autoři

VALUCHOVÁ, Soňa; Elisa DERBOVEN a Karel ŘÍHA

Vydání

Bio-protocol, Sunnyvale, Bio-protocol LLC, 2016, 2331-8325

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

Genetika a molekulární biologie

Stát vydavatele

Spojené státy

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Označené pro přenos do RIV

Ano

Kód RIV

RIV/00216224:14740/16:00093962

Organizační jednotka

Středoevropský technologický institut

DOI

https://doi.org/10.21769/BioProtoc.1775

Klíčová slova anglicky

Telomere; G-overhang; In-gel hybridization; Arabidopsis; Telomere analysis

Štítky

rivok

Změněno: 23. 3. 2017 10:15, Mgr. Eva Špillingová

Anotace

V originále

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.

Citovat

VALUCHOVÁ, Soňa; Elisa DERBOVEN a Karel ŘÍHA. Analysis of Telomeric G -overhangs by in- Gel Hybridization. Bio-protocol. Sunnyvale: Bio-protocol LLC, 2016, roč. 6, č. 7, s. nestránkováno, 9 s. ISSN 2331-8325. Dostupné z: https://doi.org/10.21769/BioProtoc.1775.

@article{1376223,
   author = {Valuchová, Soňa and Derboven, Elisa and Říha, Karel},
   article_location = {Sunnyvale},
   article_number = {7},
   doi = {https://doi.org/10.21769/BioProtoc.1775},
   keywords = {Telomere; G-overhang; In-gel hybridization; Arabidopsis; Telomere analysis},
   language = {eng},
   issn = {2331-8325},
   journal = {Bio-protocol},
   title = {Analysis of Telomeric G -overhangs by in- Gel Hybridization},
   url = {http://www.bio-protocol.org/e1775},
   volume = {6},
   year = {2016}
}

TY  - JOUR
ID  - 1376223
AU  - Valuchová, Soňa - Derboven, Elisa - Říha, Karel
PY  - 2016
TI  - Analysis of Telomeric G -overhangs by in- Gel Hybridization
JF  - Bio-protocol
VL  - 6
IS  - 7
SP  - nestránkováno
EP  - nestránkováno
PB  - Bio-protocol LLC
SN  - 23318325
KW  - Telomere
KW  - G-overhang
KW  - In-gel hybridization
KW  - Arabidopsis
KW  - Telomere analysis
UR  - http://www.bio-protocol.org/e1775
L2  - http://www.bio-protocol.org/e1775
N2  - 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.
ER  -

VALUCHOVÁ, Soňa; Elisa DERBOVEN a Karel ŘÍHA. Analysis of Telomeric G -overhangs by in- Gel Hybridization. \textit{Bio-protocol}. Sunnyvale: Bio-protocol LLC, 2016, roč.~6, č.~7, s.~nestránkováno, 9 s. ISSN~2331-8325. Dostupné z: https://doi.org/10.21769/BioProtoc.1775.

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