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 a Jiří FAJKUS

Základní údaje

Originální název

Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths

Autoři

FOJTOVÁ, Miloslava (203 Česká republika, domácí), Petr FAJKUS (203 Česká republika, domácí), Pavla SOVÁKOVÁ (203 Česká republika, domácí) a Jiří FAJKUS (203 Česká republika, garant, domácí)

Vydání

Bio-protocol, Sunnyvale, Bio-protocol LLC, 2015, 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

Kód RIV

RIV/00216224:14740/15:00088145

Organizační jednotka

Středoevropský technologický institut

DOI

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

Klíčová slova anglicky

method; telomere length; protocol

Štítky

rivok

Příznaky

Mezinárodní význam

Změněno: 27. 3. 2017 13:28, Mgr. Eva Špillingová

Anotace

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).

Návaznosti

ED1.1.00/02.0068, projekt VaV

Název: CEITEC - central european institute of technology

GA13-06595S, projekt VaV

Název: Telomery a stabilita genomu u nižších rostlin

Investor: Grantová agentura ČR, Telomery a stabilita genomu u nižších rostlin

GA13-06943S, projekt VaV

Název: Strukturní a funkční komponenty rostlinných telomer

Investor: Grantová agentura ČR, Strukturní a funkční komponenty rostlinných telomer

Citovat

FOJTOVÁ, Miloslava, Petr FAJKUS, Pavla SOVÁKOVÁ a Jiří FAJKUS. Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths. Bio-protocol. Sunnyvale: Bio-protocol LLC, 2015, roč. 5, č. 23, s. nestránkováno, 12 s. ISSN 2331-8325. Dostupné z: 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 a Jiří FAJKUS. Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths. \textit{Bio-protocol}. Sunnyvale: Bio-protocol LLC, 2015, roč.~5, č.~23, s.~nestránkováno, 12 s. ISSN~2331-8325. Dostupné z: https://dx.doi.org/10.21769/BioProtoc.1671.

Podrobný výpis o publikaci