Quantitative amplification of single-stranded DNA (QAOS) demonstrates that cdc13-1 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 (dsDNA). Single-stranded DNA (ssDNA) is first captured by annealing a tagging primer 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 QAOS to characterise ssDNA levels at three loci near the right telomere of chromosome V in budding yeast cdc13-1 mutants. Our results confirm and extend previous studies which demonstrate that when Cdc13p, a telomere-binding protein, is disabled, loci close to the telomere become single stranded whereas centromere proximal sequences do not. In contrast to an earlier model, our new results are consistent with a model in which a RAD24-dependent, 5' to 3' exonuclease moves from the telomere toward the centromere in cdc13-1 mutants. QAOS has been adapted, using degenerate tagging primers, to preferentially amplify all ssDNA sequences within samples that are primarily dsDNA. This approach may be useful for identifying ssDNA sequences associated with physiological or pathological states in other organisms.
Booth
C
C
School of Biological Sciences, University of Manchester, G38 Stopford Building, Oxford Road, Manchester M13 9PT, UK.
Griffith
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E
Brady
G
G
Lydall
D
D
D018797:Cell Cycle Proteins / Q000378:metabolism; D002503:Centromere / Q000235:genetics*; D015825:Chromosomes, Fungal / Q000235:genetics; D019926:Cyclin B / Q000235:genetics*; D017931:DNA Primers; D015342:DNA Probes; D004271:DNA, Fungal / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics; D016681:Genome, Fungal; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D016615:Telomere / Q000235:genetics*; D013696:Temperature; D013698:Templates, Genetic; D013997:Time Factors
D018797:Cell Cycle Proteins; D019926:Cyclin B; D017931:DNA Primers; D015342:DNA Probes; D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D047908:Intracellular Signaling Peptides and Proteins; C088385:RAD24 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C069930:rad9 protein