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Reversible ratiometric probe for quantitative DNA measurements.

Jörn Ueberfeld1, David R Walt

  • 1The Max Tishler Laboratory for Organic Chemistry, Department of Chemistry, Tufts University, Medford, MA 02155, USA.

Analytical Chemistry
|February 14, 2004
PubMed
Summary

We developed a reversible fluorescent DNA probe for quantifying single-stranded DNA. This probe uses ratiometric fluorescence measurements, offering a precise method for determining DNA concentrations in solution.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Analytical Chemistry

Background:

  • Accurate quantification of single-stranded DNA (ssDNA) is crucial in molecular biology and diagnostics.
  • Existing methods may have limitations in sensitivity, specificity, or ease of use.
  • Development of novel probes for ssDNA detection is an ongoing area of research.

Purpose of the Study:

  • To design and validate a novel reversible fluorescent DNA probe for quantitative ssDNA detection.
  • To utilize ratiometric fluorescence measurements for accurate concentration determination.
  • To establish a probe system independent of probe concentration for reliable quantification.

Main Methods:

  • Design of a single-stranded DNA probe with a stem-loop structure.
  • Dual labeling of the probe with two distinct fluorescent dyes (Cy3 and Cy5).

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  • Utilizing fluorescence resonance energy transfer (FRET) between dyes in the closed probe state.
  • Monitoring changes in donor and acceptor fluorescence upon target hybridization.
  • Employing ratiometric analysis (acceptor/donor intensity) for quantification.

    • Main Results:

    • The designed probe exhibits a stem-loop conformation in solution.
    • Hybridization with target ssDNA causes the probe to open, altering FRET efficiency.
    • A measurable change in fluorescence intensity ratio (acceptor/donor) correlates with target concentration.
    • The ratiometric measurement is independent of the total probe concentration used.

    Conclusions:

    • The reversible fluorescent DNA probe enables accurate and quantitative detection of ssDNA.
    • Ratiometric fluorescence measurement provides a robust method for ssDNA quantification, independent of probe amount.
    • This probe design offers a sensitive and reliable tool for ssDNA analysis in various biological applications.