ωqPCR determines absolute telomere length in kb units from single cells. Accuracy and precision of ωqPCR were assessed using 800 bp and 1600 bp synthetic telomeres inserted into plasmids, which were measured to be 819 ± 19.6 and 1590 ± 42.3 bp, respectively. This is the first telomere length measuring method verified in this way. The approach uses ω-probes, a DNA strand containing sequence information that enables: (i) hybridization with the telomere via the 3′ and 5′ ends that become opposed; (ii) ligation of the hybridized probes to circularize the ω-probes and (iii) circularized-dependent qPCR due to sequence information for a forward primer, and for a reverse primer binding site, and qPCR hydrolysis probe binding. Read through of the polymerase during qPCR occurs only in circularized ω-probes, which quantifies their number that is directly proportional to telomere length. When used in concert with information about the cell cycle stage from a single-copy gene, and ploidy, the MTL of single cells measured by ωqPCR was consistent with that obtained from large sample sizes by TRF.
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