Research Paper | Published:

Exponential Amplification of Recombinant- RNA Hybridization Probes

Bio/Technologyvolume 6pages11971202 (1988) | Download Citation

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Abstract

We have synthesized recombinant RNA molecules that function both as hybridization probes and as templates for exponential amplification by Qβ replicase. Each recombinant consists of a sequence specific for the protozoan parasite, Plasmodium falciparum, embedded within the sequence of MDV-1 RNA, which is a natural template for Qβ replicase. The probe sequence was inserted within a hairpin loop that occurs on the exterior of MDV-1 RNA. The recombinant RNAs hybridize specifically to complementary DNA, despite topological constraints on the probe domain, are replicated at the same rate as MDV-1 RNA, despite their additional length, and are able to serve as templates for the synthesis of a large number of RNA copies. A Qβ replicase reaction initiated with only 0.14 femtograms of recombinant RNA (1,000 molecules) can produce 129 nanograms of recombinant RNA product in 30 minutes. This represents a one-billion fold amplification. Our results demonstrate the feasibility of employing exponentially replicatable RNAs in bioassays, where they would serve the dual role of specific probe and amplifiable reporter.

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Affiliations

  1. Centro de Investigation sobre Ingeniería Genética y Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, 62270, Cuernavaca, Morelos, México

    • Paul M. Lizardi
    • , Cesar E. Guerra
    • , Hilda Lomeli
    •  & Isabel Tussie-Luna
  2. Department of Molecular Genetics, Public Health Research Institute, 455 First Avenue, New York, NY, 10016

    • Fred Russell Kramer

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https://doi.org/10.1038/nbt1088-1197

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