Methods for the assessment of telomere status

Most methods for examining telomere functionality have relied on measurements of telomeric DNA by hybridization or quantitative PCR. While these techniques yield measures of telomeric DNA length, they generate whole-population results. However, telomeric DNA lengths on different chromatids even in the same cell are usually heterogeneous. Also, these measurements do not reveal whether a particular telomere contains the critical minimum DNA length to be functional. Therefore, in order to gain a more complete knowledge of cellular health, an alternative method that reveals the functional status of each individual telomere is needed. Based on the fact that a dysfunctional telomere induces a DNA damage response, we developed a novel technique which combines a DNA damage marker with fluorescence in situ hybridization (FISH) of telomeric DNA on metaphase chromosomes to assess the functional status of individual telomeres. This technique reveals not only whether the telomeric DNA in each chromatid is significantly shortened, but also whether the telomere has induced a DNA damage response, i.e., has become dysfunctional. We describe here in detail the protocols for simultaneous assessment of telomere length and functionality.