Inventing molecular beacons

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

The invention of molecular beacons followed a rather circuitous route. Our laboratory had been studying the remarkable mechanism of replication of the single-stranded genomic ribonucleic acid (RNA) of bacteriophage Qβ, a virus that infects <italic>Escherichia coli</italic>. When a few molecules of Qβ RNA are incubated in a test tube with the viral RNA-directed RNA polymerase, Qβ replicase, millions of copies of each Qβ RNA molecule are generated in only a few minutes by exponential amplification, without primers and without thermal cycling. Unfortunately, Qβ replicase is so specific for the particular sequences and structures present in Qβ RNA that it ignores almost all other nucleic acid molecules, disappointing scientists who would use its extraordinary amplification characteristics to generate large amounts of any desired RNA in vitro. However, our laboratory discovered that if a heterologous RNA sequence is inserted into an appropriate site within Midivariant RNA (MDV-1), which is a naturally occurring small RNA isolated from Qβ-infected <italic>E. coli</italic> that possesses the sequences and structures required for replication, the resulting “recombinant RNA” can be amplified exponentially by incubation with Qβ replicase. This discovery enabled the design of recombinant RNAs that contained inserted hybridization probe sequences, which were employed in the earliest real-time exponential amplification assays, and the use of which, paradoxically, led to the invention of molecular beacons. Spurred by the emergence of the pernicious infectious agent human immunodeficiency virus (HIV)-1, which is present in as few as 1 in 100,000 peripheral blood mononuclear cells in infected asymptomatic individuals, we developed an assay that was designed to use the exponential amplification of recombinant RNA hybridization probes to measure the number of HIV-1 target molecules present in clinical samples.

Original languageEnglish (US)
Title of host publicationThe PCR Revolution
Subtitle of host publicationBasic Technologies and Applications
PublisherCambridge University Press
Pages19-47
Number of pages29
ISBN (Electronic)9780511818974
ISBN (Print)9780521882316
DOIs
StatePublished - Jan 1 2009

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All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Kramer, F. R., Marras, S. A. E., & Tyagi, S. (2009). Inventing molecular beacons. In The PCR Revolution: Basic Technologies and Applications (pp. 19-47). Cambridge University Press. https://doi.org/10.1017/CBO9780511818974.005