Quantitative amplification of single-stranded DNA (QAOS) demonstrates thatcdc13-1 mutants generate ssDNA in a telomere to centromere direction

We have developed a method that allows quantitative amplification of single -stranded DNA (QAOS) in a sample that is primarily double-stranded DNA (dsD NA). Single-stranded DNA (ssDNA) is first captured by annealing a tagging p rimer at low temperature. Primer extension follows to create a novel, ssDNA -dependent, tagged molecule that can be detected by PCR. Using QAOS levels of between 0.2 and 100% ssDNA can be accurately quantified. We have used QA OS to characterise ssDNA levels at three loci near the right telomere of ch romosome V in budding yeast cdc13-1 mutants. Our results confirm and extend previous studies which demonstrate that when Cdc13p, a telomere-binding pr otein, is disabled, loci close to the telomere become single stranded where as centromere proximal sequences do not. In contrast to an earlier model, o ur new results are consistent with a model in which a RAD24-dependent, 5' t o 3' exonuclease moves from the telomere toward the centromere in cdc13-1 m utants. QAOS has been adapted, using degenerate tagging primers, to prefere ntially amplify all ssDNA sequences within samples that are primarily dsDNA . This approach may be useful for identifying ssDNA sequences associated wi th physiological or pathological states in other organisms.