Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths

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).