Telomeres and Replicative Senescence

Telomere length measurement can be used both to monitor the proliferation of long-term cultures of somatic cells as well as to determine the replicative history of in vivo-derived cells. The most frequently used technique for telomere length measurement is Southern hybridization (1 , 4 ). The method consists of isolating total genomic DNA, digesting the DNA with restriction enzymes so as to isolate the undigested telomere restriction fragments (TRFs), and separating these fragments by gel electrophoresis. The DNA is denatured and transferred from the gel to a membrane or filter, and the DNA samples are then hybridized to radiolabeled complementary probe. However, when blotting TRF DNA to the membrane, differential transfer may occur owing to inefficient transfer of larger fragments of DNA (>10 kb) to a membrane. As the mean length of the TRF is based on the assumption that the amount of telomeric DNA (TTAGGG repeats) in a given TRF is proportional to the length (3 , 4 ), this would lead to possible error in calculating the mean length of the telomeres. The method that we present here avoids these potential problems by eliminating the membrane blot step altogether and probing the gel directly.