Use of SAM2® biotin capture membrane in microarrayed compound screening (μARCS) format for nucleic acid polymerization assays

Xiaoling Xuei, Caroline A. David, Tim R. Middleton, Ben Lim, Ron Pithawalla, Chih Ming Chen, Rakesh L. Tripathi, David J. Burns, Usha Warrior

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

Microarrayed compound screening format (μARCS) is a novel high-throughput screening technology that uses agarose matrices to integrate various biochemical or biological reagents in the assay. To evaluate the feasibility of using the μARCS technology for nucleic acid polymerization assays, the authors developed HIV reverse transcription (RT) and E1-dependent human papillomavirus (HPV) replication assays in this format. HIV RT is an RNA-dependent DNA polymerase, whereas HPV E1 is a DNA helicase. To ensure the efficient capture of the nucleic acid polymerization reaction and to minimize the nonspecific binding, the authors used a SAM2® biotin capture membrane in the assay. In both studies, the nucleic acid substrate was biotinylated on one end and was bound to the SAM2® membrane. A low melting-point agarose gel containing the rest of the reaction components was first placed on a polystyrene sheet spotted with compounds to allow passive diffusion of the compounds into the gel. The gel was removed from the compound sheet and applied to the SAM2® membrane with the immobilized nucleic acid template to initiate the polymerization. After the incubation, the membrane was washed with a high-salt buffer and exposed for imaging. Potential inhibitors can be seen as white spots on a dark background. The sensitivity for the known inhibitors appears to be comparable in μARCS as in a traditional 96-well plate assay. The methodology described in this paper further expands the applications of μARCS technology.

Original languageEnglish (US)
Pages (from-to)273-282
Number of pages10
JournalJournal of Biomolecular Screening
Volume8
Issue number3
DOIs
StatePublished - Jun 1 2003

Keywords

  • μARCS
  • High-throughput screening
  • Nucleic acid polymerization assays
  • SAM®

ASJC Scopus subject areas

  • Analytical Chemistry
  • Clinical Biochemistry
  • Biotechnology
  • Biochemistry
  • Molecular Biology

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    Xuei, X., David, C. A., Middleton, T. R., Lim, B., Pithawalla, R., Chen, C. M., Tripathi, R. L., Burns, D. J., & Warrior, U. (2003). Use of SAM2® biotin capture membrane in microarrayed compound screening (μARCS) format for nucleic acid polymerization assays. Journal of Biomolecular Screening, 8(3), 273-282. https://doi.org/10.1177/1087057103008003005