Aging Aging

Telomeres and Replicative Senescence 634Telomeres and Replicative SenescenceHector F. Valenzuela and Rita B. Effros1. IntroductionTelomere length measurement can be used both to monitor the proliferationof long-term cultures of somatic cells as well as to determine the replicativehistory of in vivo-derived cells. The most frequently used technique for telomerelength measurement is Southern hybridization (1,2). The method consistsof isolating total genomic DNA, digesting the DNA with restriction enzymesso as to isolate the undigested telomere restriction fragments (TRFs), and separatingthese fragments by gel electrophoresis. The DNA is denatured and transferredfrom the gel to a membrane or filter, and the DNA samples are thenhybridized to radiolabeled complementary probe. However, when blotting TRFDNA to the membrane, differential transfer may occur owing to inefficienttransfer of larger fragments of DNA (>10 kb) to a membrane. As the meanlength of the TRF is based on the assumption that the amount of telomericDNA (TTAGGG repeats) in a given TRF is proportional to the length (3,4), thiswould lead to possible error in calculating the mean length of the telomeres.The method that we present here avoids these potential problems by eliminatingthe membrane blot step altogether and probing the gel directly.The following protocol has been refined for measuring telomeric DNAlength from human cells. Similar protocols can be adapted to measuretelomere lengths in cells from other species. However, researchers shouldadjust the probe sequence for hybridization (not all species have the sametelomere sequence) and optimize the restriction enzymes to obtain TRF withinthe resolvable molecular weight range of the gel because some species mayhave extremely long telomeres. For more information regarding telomeres, wesuggest reading Kipling’s The Telomeres (5).From: Methods in Molecular Medicine, Vol. 38: Aging Methods and ProtocolsEdited by: Y. A. Barnett and C. R. Barnett © Humana Press Inc., Totowa, NJ63