Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Nucleic Acids Res. 2001 Nov 1;29(21):4414-22.

Quantitative amplification of single-stranded DNA (QAOS) demonstrates that cdc13-1 mutants generate ssDNA in a telomere to centromere direction.

Author information

  • 1School of Biological Sciences, University of Manchester, G38 Stopford Building, Oxford Road, Manchester M13 9PT, UK.

Abstract

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.

PMID:
11691929
[PubMed - indexed for MEDLINE]
PMCID:
PMC60175
Free PMC Article

Images from this publication.See all images (4)Free text

Figure 1
Figure 2
Figure 3
Figure 4
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for HighWire Icon for PubMed Central
    Loading ...
    Write to the Help Desk