Absolute Length Distribution of Human Telomeres with Single-Molecule Techniques

Human telomeres exhibit progressive shortening with each replication cycle. This phenomenon plays a critical role in the onset of senescence and the development of cancers. The measurement of absolute telomere length (TL) is not only serving as a marker of aging, but also holds substantial medical relevance. However, current TL measurement technologies face significant challenges, including limited precision, inability to resolve TL heterogeneity or distinguish telomeric signals from interstitial telomeric sequences (ITS), and data inconsistency. Single-molecule mechanical techniques have shown promise in manipulating DNA and providing precise contour length measurements of DNA, making them suitable for assessing TL quantitatively. In this study, we developed a method, named single-molecule terminal restriction fragment (smTRF) analysis, for measuring telomeres at single-molecule resolution. We applied smTRF to seven human cancer cell lines and successfully determined TL ranging from a few to tens of kilobases, highlighting the versatility and high-fidelity performance of the smTRF assay. The smTRF data were validated against results from standard TRF, qPCR, and Q-FISH analysis, demonstrating well agreement and confirming the assay's reliability in the measurement of average TL. To further test the robustness of smTRF, we measured TL distribution profiles for 48 individuals, establishing the smTRF assay as a reliable tool for the accurate and precise measurement of human TLs. The comprehensive telomere profiles obtained via the smTRF assay promise to provide in-depth insights into public health research, particularly in the study of aging, where TL serves as a critical biomarker.