Terminal Restriction Fragments (TRF) Method to Analyze Telomere
Lengths

J 2015

Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths

FOJTOVÁ, Miloslava, Petr FAJKUS, Pavla SOVÁKOVÁ and Jiří FAJKUS

Basic information

Original name

Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths

Authors

FOJTOVÁ, Miloslava (203 Czech Republic, belonging to the institution), Petr FAJKUS (203 Czech Republic, belonging to the institution), Pavla SOVÁKOVÁ (203 Czech Republic, belonging to the institution) and Jiří FAJKUS (203 Czech Republic, guarantor, belonging to the institution)

Edition

Bio-protocol, Sunnyvale, Bio-protocol LLC, 2015, 2331-8325

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

Genetics and molecular biology

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

RIV identification code

RIV/00216224:14740/15:00088145

Organization unit

Central European Institute of Technology

DOI

http://dx.doi.org/10.21769/BioProtoc.1671

Keywords in English

method; telomere length; protocol

Abstract

V originále

Chromosome ends - telomeres - are a focus of intensive research due to their importance for the maintenance of chromosome stability. Their shortening du e to incomplete replication functions as a molecular clock counting the number of cell divi sions, and ultimately results in cell-cycle arrest and cellular senescence. Determination of te lomere lengths is an essential approach in telomere biology for research and diagnostic applications. Term inal Restriction Fragments (TRF) analysis is the oldest approach to analyze telomere lengths and remains the “gold standard” even in current studies. This technique relies on the fact that repeated minisatellite telomeric units do not contain target sites for restri ction enzymes. Consequently, telomeres remain in relatively long fragments (TRF), whereas the genomic DNA is digested into short pieces. Fragments of telomeric DNA are then visualized by hybridization with radioactively labeled telomeric probe. As TRF include besides telomeres also a short region of telomere-associated DNA up to the first restriction site, resul ts are slightly shifted towards higher TRFs values. Therefore, the use of frequent cutters or their mixtures is recommended to minimize this difference. Moreover, by using TRF analysis it is possible to distinguish genuine (terminal) telomeres from interstitial telomeric repeats (ITR) (Richa rds and Ausubel, 1988). In this approach, BAL31 digestion is first applied on high molec ular weight DNA. The enzyme progressively degrades linear DNA from its ends. The degrad ed DNA is then digested with one or more restriction enzymes and fragments are separated by gel electrophoresis. After blotting, membranes are probed with either a terminal marker sequence or telomeric sequence. Genuine TRF can be distinguished from ITR due to their progres sive shortening with increasing BAL31 digestion time, while ITR are BAL31-resistan t. The TRF BAL31 digestion pattern at the time zero indicates the approximate telomere lengths (Fajkus et al. , 2005).

Links

ED1.1.00/02.0068, research and development project

Name: CEITEC - central european institute of technology

GA13-06595S, research and development project

Name: Telomery a stabilita genomu u nižších rostlin

Investor: Czech Science Foundation

GA13-06943S, research and development project

Name: Strukturní a funkční komponenty rostlinných telomer

Investor: Czech Science Foundation

Citovat

FOJTOVÁ, Miloslava, Petr FAJKUS, Pavla SOVÁKOVÁ and Jiří FAJKUS. Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths. Bio-protocol. Sunnyvale: Bio-protocol LLC, 2015, vol. 5, No 23, p. nestránkováno, 12 pp. ISSN 2331-8325. Available from: https://dx.doi.org/10.21769/BioProtoc.1671.

@article{1354106,
   author = {Fojtová, Miloslava and Fajkus, Petr and Sováková, Pavla and Fajkus, Jiří},
   article_location = {Sunnyvale},
   article_number = {23},
   doi = {http://dx.doi.org/10.21769/BioProtoc.1671},
   keywords = {method; telomere length; protocol},
   language = {eng},
   issn = {2331-8325},
   journal = {Bio-protocol},
   title = {Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths},
   url = {http://www.bio-protocol.org/pdf/Bio-protocol1671.pdf},
   volume = {5},
   year = {2015}
}

TY  - JOUR
ID  - 1354106
AU  - Fojtová, Miloslava - Fajkus, Petr - Sováková, Pavla - Fajkus, Jiří
PY  - 2015
TI  - Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths
JF  - Bio-protocol
VL  - 5
IS  - 23
SP  - nestránkováno
EP  - nestránkováno
PB  - Bio-protocol LLC
SN  - 23318325
KW  - method
KW  - telomere length
KW  - protocol
UR  - http://www.bio-protocol.org/pdf/Bio-protocol1671.pdf
L2  - http://www.bio-protocol.org/pdf/Bio-protocol1671.pdf
N2  - Chromosome ends - telomeres - are a focus of intensive research due to their importance for the maintenance of chromosome stability. Their shortening du e to incomplete replication functions as a molecular clock counting the number of cell divi sions, and ultimately results in cell-cycle arrest and cellular senescence. Determination of te lomere lengths is an essential approach in telomere biology for research and diagnostic applications. Term inal Restriction Fragments (TRF) analysis is the oldest approach to analyze telomere lengths and remains the “gold standard” even in current studies. This technique relies on the fact that repeated minisatellite telomeric units do not contain target sites for restri ction enzymes. Consequently, telomeres remain in relatively long fragments (TRF), whereas the genomic DNA is digested into short pieces. Fragments of telomeric DNA are then visualized by hybridization with radioactively labeled telomeric probe. As TRF include besides telomeres also a short region of telomere-associated DNA up to the first restriction site, resul ts are slightly shifted towards higher TRFs values. Therefore, the use of frequent cutters or their mixtures is recommended to minimize this difference. Moreover, by using TRF analysis it is possible to distinguish genuine (terminal) telomeres from interstitial telomeric repeats (ITR) (Richa rds and Ausubel, 1988). In this approach, BAL31 digestion is first applied on high molec ular weight DNA. The enzyme progressively degrades linear DNA from its ends. The degrad ed DNA is then digested with one or more restriction enzymes and fragments are separated by gel electrophoresis. After blotting, membranes are probed with either a terminal marker sequence or telomeric sequence. Genuine TRF can be distinguished from ITR due to their progres sive shortening with increasing BAL31 digestion time, while ITR are BAL31-resistan t. The TRF BAL31 digestion pattern at the time zero indicates the approximate telomere lengths (Fajkus et al. , 2005).
ER  -

FOJTOVÁ, Miloslava, Petr FAJKUS, Pavla SOVÁKOVÁ and Jiří FAJKUS. Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths. \textit{Bio-protocol}. Sunnyvale: Bio-protocol LLC, 2015, vol.~5, No~23, p.~nestránkováno, 12 pp. ISSN~2331-8325. Available from: https://dx.doi.org/10.21769/BioProtoc.1671.

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