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Related Experiment Videos

Fluorescently labeled model DNA sequences for exonucleolytic sequencing.

Z Földes-Papp1, B Angerer, P Thyberg

  • 1Department of Medical Biophysics, MBB, Karolinska Institute, S-17177 Stockholm, Sweden. Zeno.Foldes-Papp@kfunigraz.ac.at

Journal of Biotechnology
|March 21, 2001
PubMed
Summary
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This study demonstrates enzyme-catalyzed DNA labeling with fluorescent dyes using PCR. Optimized synthetic DNA constructs allow precise dye incorporation for exonucleolytic sequencing applications.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Enzyme-catalyzed labeling of DNA with fluorescent dyes is crucial for various molecular biology applications.
  • Current methods face challenges in achieving precise and efficient incorporation of fluorophore-bearing analogs into DNA templates.

Purpose of the Study:

  • To develop and optimize methods for low-density labeling of DNA with fluorescent dyes using enzymatic approaches.
  • To evaluate the efficiency of different fluorophore-bearing deoxynucleotide triphosphates (dNTPs) incorporation.
  • To demonstrate the utility of synthetic DNA constructs for precise fluorescent labeling and exonucleolytic sequencing.

Main Methods:

  • Polymerase Chain Reaction (PCR) using Taq DNA polymerase for labeling natural DNA templates with dye-dNTPs.

Related Experiment Videos

  • Partial substitution of natural dNTPs with fluorophore-bearing analogs (rhodamine-green-5-dUTP, tetramethylrhodamine-4-dUTP, Cy5-dCTP).
  • Exonucleolytic degradation using T7 DNA polymerase to determine incorporation efficiency.
  • PCR using a thermostable 3'-->5' exonuclease-deficient Tgo DNA polymerase mutant for labeling synthetic DNA constructs with dye-dNTPs (rhodamine-green-X-dUTP, tetramethylrhodamine-6-dUTP, Cy5-dCTP).
  • Fluorescence correlation spectroscopy (FCS) to determine catalytic cleavage constants and quantify incorporated nucleotides.
  • Re-sequencing methodologies (mobility-shift gel electrophoresis, reversion-PCR) for confirming covalent and base-specific incorporation.

    • Main Results:

    • The covalent incorporation efficiency of dye-dNTPs into natural DNA decreased in the order: rhodamine-green-5-dUTP > tetramethylrhodamine-4-dUTP > Cy5-dCTP.
    • Exonucleolytic degradation of labeled natural DNA by T7 DNA polymerase was characterized by catalytic cleavage constants between 0.5 and 1.5 s⁻¹.
    • Synthetic DNA constructs allowed for complete substitution and precise positioning of dye-dNTPs, with six out of six possible incorporations achieved for rhodamine-green-X-dUTP and tetramethylrhodamine-6-dUTP.
    • Novel re-sequencing analysis confirmed the covalent and base-specific incorporation of fluorescent nucleotides.

    Conclusions:

    • Enzyme-catalyzed PCR enables efficient low-density labeling of DNA with fluorescent dyes.
    • Synthetic DNA constructs offer a powerful platform for creating tailor-made nucleic acids with precisely positioned fluorescent labels.
    • The developed methodology and analysis techniques are valuable for advancing exonucleolytic sequencing and other DNA analysis applications.