DNA polymerase extension assay for influenza virus endonuclease

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

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536 231, 536 243, C12Q 168

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056609892

ABSTRACT:
An assay for the influenza virus endonuclease has been developed which involves DNA polymerase-catalyzed extension of the viral endonuclease cleavage product using labeled nucleotides and a DNA template containing a 3' region complementary to the product joined to a 5' region consisting of repeated residues. The DNA polymerase coupled assay does not involve gel electrophoretic separation and is amenable to high volume screening of potential inhibitors. Another key feature of the assay is that it is sensitive enough to detect 200 attomoles of product.

REFERENCES:
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Tomassini et al. Antimicrobial Agents and Chemotherapy 38(12):2827-2837 (1994).
The Amersham Catalog p. 40 (1993).
James M. Clark, "Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences", Research Triangle Park, NC 27709, USA. vol. 16 No. 20, 1988.
Stephen J. Plotch, et al. "A Unique Cap(m7GpppXm)-Dependent Influenza Virion Endonuclease Cleaves Capped RNAs to Generate the Primers That Initiate Viral RNA Transcription", Cell, vol. 23, 847-858, Mar. 1981.
Stephen J. Plotch, et al. "Influenza Virion Transcriptase: Synthesis In Vitro of Large, Polyadenylic Acid-Containing Complementary RNA" Journal of Virology, Jan. 1977, pp. 24-34, vol. 21, No. 1.
Stanley Tabor et al. "Selective Inactivation of the Exonuclease Activity of Bacteriophage T7 DNA Polymerase by in Vitro Mutagenesis*" The Journal of Biological Chemistry, vol. 264, No. 11, Issue Apr. 15, pp. 6447-6458, 1989.
J. Tomassini, et al. "Inhibition of Cap(m7GpppXm)-Dependent Endonuclease of Influenza Virus by 4-Substituted 2,4-Dioxobutanoic Acid Compounds" Antimicrobial Agents and Chemotherapy, Dec. 1994, pp. 2827-2837.

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