Simple, rapid, quantitative, and sensitive detection of telomere repeats in cell lysate by a hybridization protection assay

Background: Detection of telomere repeats by Southern hybridization of geno mic DNA is time consuming, and the reading of a mean terminal restriction f ragment (TRF) length from a smear pattern of an autoradiogram can be inaccu rate. We developed a hybridization protection assay (HPA) for telomere repe ats. Methods: We heated 5 mu L of DNA solution or 10 mu L, of cell or tissue lys ate at 95 degrees C for 5 min, mixed it with 100 mu L of hybridization solu tion containing 3 x 10(6) relative light units of acridinium ester-labeled probe, and incubated the mixture for 20 min at 60 degrees C. We then added 300 mu L of selection buffer and incubated the mixture for 10 min at 60 deg rees C to differentially hydrolyze unhybridized probe. Chemiluminescence wa s 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 eq uivalents of lysate. To normalize the amount of DNA in lysate, the amount o f Alu sequence was measured by HPA. A ratio of telomere to Abl (TA ratio) = 0.01 corresponded to similar to 2 kbp of mean TRF length determined by Sou thern 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 assa y required similar to 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 me thod is simple, rapid, quantitative, sensitive, and applicable to the measu rement of telomere repeats in clinical samples such as needle biopsy specim en or as few as 1000 cells in body fluid or washings. (C) 1999 American Ass ociation for Clinical Chemistry.