Published: Vol 5, Iss 22, Nov 20, 2015 DOI: 10.21769/BioProtoc.1656 Views: 12118
Reviewed by: HongLok LungVanesa Olivares-IllanaAnonymous reviewer(s)
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Telomerase Repeated Amplification Protocol (TRAP)
Ilgen Mender and Jerry W. Shay
Nov 20, 2015 21644 Views
Telomere Restriction Fragment (TRF) Analysis
Ilgen Mender and Jerry W. Shay
Nov 20, 2015 21094 Views
Abstract
Telomerase maintains telomeric DNA in eukaryotes during early developments, ~90% of cancer cells and some proliferative stem like cells. Telomeric repeats at the end of chromosomes are associated with the shelterin complex. This complex consists of TRF1, TRF2, Rap1, TIN2, TPP1, POT1 which protect DNA from being recognized as DNA double-stranded breaks. Critically short telomeres or impaired shelterin proteins can cause telomere dysfunction, which eventually induces DNA damage responses at the telomeres. DNA damage responses can be identified by antibodies to 53BP1, gammaH2AX, Rad17, ATM, and Mre11. DNA damage foci at uncapped telomeres are referred to as Telomere dysfunction-Induced Foci (TIFs) (de Lange, 2005; Takai et al., 2003).
The TIF assay is based on the co-localization detection of DNA damage by an antibody against DNA damage markers, such as gamma-H2AX, and telomeres using an antibody against one of the shelterin proteins such as TRF2 (Takai et al., 2003; de Lange, 2002; Karlseder et al., 1999). The method we describe here can be used in normal human and cancer cells.
Other commonly used methods-Telomere Restriction Fragment (TRF) Analysis (Mender and Shay, 2015b) and Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015a)- in telomere biology can be found by clicking on the indicated links.
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Acknowledgments
Some of these protocols were adapted from previously published studies. We thank Zeliha Gunnur Dikmen for her help in acquisition of TRAP gel and Abhijit Bugde from the Live Cell Imaging Facility at UT Southwestern for his assistance with the imaging and analysis part of Telomere dysfunction Induced Foci (TIF) analysis.
References
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Copyright
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Mender, I. and Shay, J. W. (2015). Telomere Dysfunction Induced Foci (TIF) Analysis. Bio-protocol 5(22): e1656. DOI: 10.21769/BioProtoc.1656.
Category
Cancer Biology > Replicative immortality > Cell biology assays
Cancer Biology > Replicative immortality > Cell biology assays
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