凝胶杂交分析端粒的G悬突

Sona Valuchova; Elisa Derboven; Karel Riha

doi:10.21769/BioProtoc.1775

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

Analysis of Telomeric G-overhangs by in-Gel Hybridization

凝胶杂交分析端粒的G悬突

SV Sona Valuchova ^*

ED Elisa Derboven ^*

KR Karel Riha email

(*contributed equally to this work) 发布: 2016年04月05日第6卷第7期 DOI: 10.21769/BioProtoc.1775 浏览次数: 8444

评审: Tie LiuShyam SolankiTzvetina Brumbarova

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Genetics, featuring study using the protocol.

Oct 2014

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

基于微孔板的拟南芥硝酸盐转运蛋白2.1表达监测系统——利用荧光素酶报告基因

Yoshiaki Ueda and Shuichi Yanagisawa

2024年12月05日 1042 阅读

利用分裂可选择标记的植物转化新型基因叠加方法

Guoliang Yuan [...] Xiaohan Yang

2025年02月20日 944 阅读

三种可可属植物的DNA提取优化方法

Angie F. Riascos-España [...] Pedro A. Velasquez-Vasconez

2025年05月05日 1089 阅读

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 (端粒)

G-overhang (G overhang)

In-gel hybridization (在凝胶杂交)

Arabidopsis (拟南芥)

Telomere analysis (端粒长度的分析)

Materials and Reagents

Plastic wrap (common food wrap or Fisherbrand^TM Clear Plastic Wrap) (Thermo Fisher Scientific, catalog number: 22-305654 )
Whatman^® papers (Sigma-Aldrich, catalog number: 3030917 )
GeneJET Plant Genomic DNA Purification Kit (Life Technologies, catalog number: K0792 )
Note: Currently, it is “Thermo Fisher Scientific, catalog number: K0792”.
T4 DNA polymerase (NEB, catalog number: M0203S )
NEB buffer #2 (NEB, catalog number: B7002S )
dNTPs (Life Technologies, catalog number: R0181 )
Note: Currently, it is “Thermo Fisher Scientific, catalog number: R0181”.
Isopropanol
Absolute ethanol (EtOH)
HindIII (Thermo Fisher Scientific, catalog number: ER0501 )
3 M NaOAc (pH 5.2)
DNA Gel Loading Dye (6x) (Life Technologies, catalog number: R0611 )
Note: Currently, it is “Thermo Fisher Scientific, catalog number: R0611”.
PeqGold Universal agarose (VWR, catalog number: 732-2789 )
Ethidium Bromide (EtBr) solution (1%) (Applichem, catalog number: A1152 )
T4 polynucleotide kinase (PNK) (10 U/µl) (Thermo Fisher Scientific, catalog number: EK0031 )
Custom oligonucleotide probe, (TA3C3)4 or (TA3C3)3 (10 pmol/µl) (Sigma-Aldrich)
γ³²P-ATP (> 6,000 Ci/mmol) (HARTMANN ANALYTIC GmbH, catalog number: SRP-501 )
QIAquick Nucleotide Removal Kit (QIAGEN, catalog number: 28304 )
Tris
Acetic acid
EDTA (pH 8.0)
NaCl
Sodium citrate (pH 7.0)
SDS
Na-phosphate buffer (pH 7.2)
BSA
Tris-Cl (pH 8)
1x TAE (see Recipes)
20x SSC (see Recipes)
Hybridisation buffer (see Recipes)
Wash solution-1 (see Recipes)
Wash solution-2 (see Recipes)
Denaturation solution (see Recipes)
Neutralization solution (see Recipes)

Equipment

Eppendorf^TM ThermoMixer^TM C
Centrifuge (Eppendorf AG)
Horizontal Midi Gel Electrophoresis system (PerfectBlue Gel System Midi S, Peqlab)
Gel Doc^TM XR+ System (Bio-Rad Laboratories)
Gel dryer (Bio-Rad Laboratories, model: 583 )
Imaging plate (Fujifilm, catalog number: BAS-IP MS 2025 )
Note: Currently, it is “GE Healthcare, catalog number: BAS-IP MS 2025”.
Vacuum pump (Gardner Denver Thomas GmbH, ILMVAC, catalog number: 412711 )
Water bath with shaking
Pharos FX Plus Molecular Imager (Bio-Rad Laboratories)
PC with an image analysis software (e.g., Image Lab from Bio-Rad Laboratories)

Software

Image Lab 5.1 (Bio-Rad Laboratories) (freely available at http://www.bio-rad.com/en-us/product/image-lab-software)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Valuchova, S., Derboven, E. and Riha, K. (2016). Analysis of Telomeric G-overhangs by in-Gel Hybridization. Bio-protocol 6(7): e1775. DOI: 10.21769/BioProtoc.1775.