Simple, Rapid, Quantitative, and Sensitive Detection of Telomere Repeats in Cell Lysate by a Hybridization Protection Assay

摘要

Background: Detection of telomere repeats by Southern hybridization of genomic DNA is time consuming, and the reading of a mean terminal restriction fragment (TRF) length from a smear pattern of an autoradiogram can be inaccurate. We developed a hybridization protection assay (HPA) for telomere repeats.Methods: We heated 5 μL of DNA solution or 10 μL of cell or tissue lysate at 95 °C for 5 min, mixed it with 100 μL of hybridization solution containing 3 × 106 relative light units of acridinium ester-labeled probe, and incubated the mixture for 20 min at 60 °C. We then added 300 μL of selection buffer and incubated the mixture for 10 min at 60 °C to differentially hydrolyze unhybridized probe. Chemiluminescence was measured for 2 s per tube.Results: The amount of telomere repeats was assayed by HPA within linearity from 10 to 3000 ng of purified genomic DNA or from 1000 to 100 000 cell equivalents of lysate. To normalize the amount of DNA in lysate, the amount of Alu sequence was measured by HPA. A ratio of telomere to Alu (TA ratio) = 0.01 corresponded to ～2 kbp of mean TRF length determined by Southern blotting in cultured fibroblast and colorectal tissue samples. The TA ratio decreased from 0.06 to 0.02 with increasing division age from 30 to 90 population doubling levels of cultured human fetal fibroblasts. The assay required ～45 min from collection of cell or tissue samples.Conclusions: The amount of telomere repeats was quantitatively measured by HPA in 10 ng of sheared genomic DNA or in the lysate of 1000 cells. This method is simple, rapid, quantitative, sensitive, and applicable to the measurement of telomere repeats in clinical samples such as needle biopsy specimen or as few as 1000 cells in body fluid or washings.