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Related Experiment Videos

A novel method for DNA molecular counting.

K Hirano1, T Fukami

  • 1Tsukuba Research Laboratory, Hamamatsu Photonics K.K., 5-9-2 Tokodai, Tsukuba City, Ibaraki 300-2635, Japan.

Nucleic Acids Symposium Series
|August 9, 2003
PubMed
Summary
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This study introduces a novel capillary-plate method for precise DNA molecule counting. This technique enables accurate quantification of low DNA template concentrations through fluorescence detection in micro-channels.

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Analytical Chemistry

Background:

  • Accurate quantification of DNA molecules is crucial for various biological and diagnostic applications.
  • Existing methods may face limitations in sensitivity and precision, especially at very low template concentrations.

Purpose of the Study:

  • To develop and validate a new method for counting DNA molecules using capillary-plates.
  • To assess the sensitivity and linearity of the method for quantifying low DNA template concentrations.

Main Methods:

  • Development of capillary-plates with numerous parallel glass-capillary channels.
  • Utilizing polymerase chain reaction (PCR) with serial dilutions of DNA templates and Hoechst 33258 dye.
  • Sealing plates with silicone rubber and performing 40 PCR cycles.

Related Experiment Videos

  • Analyzing fluorescence intensity using fluorescence microscopy.

    • Main Results:

    • Increased fluorescence (approximately threefold) observed in some channels at template concentrations of 1 fM or lower.
    • No significant fluorescence detected in the absence of DNA template.
    • A linear relationship between the proportion of fluorescent channels and template concentrations (0.1-1,000 aM) was established, following Poisson probabilities.
    • Demonstrated amplification of single DNA templates within the capillary channels.

    Conclusions:

    • The capillary-plate method allows for the amplification of single DNA templates.
    • Accurate quantification of low DNA template numbers is achievable by counting positive fluorescent channels.
    • This method offers a sensitive and precise approach for DNA molecule counting.