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Related Experiment Videos

Polymerase activities and RNA structures in the atomic force microscope.

H G Hansma1, R Golan, W Hsieh

  • 1Department of Physics, University of California, Santa Barbara, Santa Barbara, California 93106-9530, USA. hhansma@physics.ucsb.edu

Journal of Structural Biology
|November 2, 1999
PubMed
Summary
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Atomic force microscopy (AFM) enables novel assays for DNA and RNA polymerases by visualizing reaction products. This technique provides rapid, semiquantitative activity estimates for enzymes like Taq polymerase and E. coli RNAP.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Nanotechnology

Background:

  • Polymerases are crucial enzymes for DNA replication and RNA transcription.
  • Accurate assays are needed to quantify polymerase activity for research and diagnostics.
  • Atomic Force Microscopy (AFM) offers high-resolution imaging of biological molecules.

Purpose of the Study:

  • To develop novel polymerase assays utilizing Atomic Force Microscopy (AFM).
  • To enable rapid, semiquantitative assessment of DNA and RNA polymerase activities.
  • To compare AFM-based assays with traditional methods like agarose gel electrophoresis.

Main Methods:

  • Utilized AFM to image nucleic acid structures resulting from polymerase reactions.
  • Assayed DNA polymerases (Sequenase, Taq, AMV reverse transcriptase) using φX-174 virion replication.

Related Experiment Videos

  • Assayed RNA polymerases (E. coli RNAP) via transcription of a rolling circle DNA template.
  • Analyzed RNA structures, including ribozyme concatamer and high-purine RNA.

    • Main Results:

    • AFM successfully visualized reaction products, enabling polymerase activity estimation.
    • AFM provided rapid, semiquantitative data for various DNA and RNA polymerases.
    • AFM outperformed agarose gel electrophoresis in cases of DNA aggregation.
    • Observed varied conformations and stretching in synthesized single-stranded RNA molecules.

    Conclusions:

    • AFM is a powerful tool for developing novel polymerase activity assays.
    • AFM offers advantages over conventional methods, particularly for aggregated samples.
    • The technique allows for structural characterization of synthesized nucleic acids, revealing differences between RNA types.