Author: ["Paul M. Lizardi","Cesar E. Guerra","Hilda Lomeli","Isabel Tussie-Luna","Fred Russell Kramer"]
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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.Cite this article
Lizardi, P., Guerra, C., Lomeli, H. et al. Exponential Amplification of Recombinant- RNA Hybridization Probes. Nat Biotechnol 6, 1197–1202 (1988). https://doi.org/10.1038/nbt1088-1197 