Specific detection of DNA and RNA targets using a novel isothermal nucleic acid amplification assay based on the formation of a three-way junction structure

Abstract

The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets. The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure. One probe (template for the RNA signal) contains a non-functional single-stranded T7 RNA polymerase promoter sequence. This promoter sequence is made double-stranded (hence functional) by DNA polymerase, allowing T7 RNA polymerase to generate a target-dependent RNA signal which is measured by an enzyme-linked oligosorbent assay (ELOSA). The sequence of the RNA signal is always the same, regardless of the original target sequence. The SMART assay was successfully tested in model systems with several single-stranded synthetic targets, both DNA and RNA. The assay could also detect specific target sequences in both genomic DNA and total RNA from Escherichia coli. It was also possible to generate signal from E.coli samples without prior extraction of nucleic acid, showing that for some targets, sample purification may not be required. The assay is simple to perform and easily adaptable to different targets.

Evaluation of an Isothermal Signal Amplification Method for Rapid Detection of Methicillin-Resistant Staphylococcus aureus from Patient-Screening Swabs

“…The CytAMP assay is based on isothermal signalmediated amplification of RNA technology (SMART) (12,24). Two targetspecific single-stranded oligonucleotide probes (the "template" probe and the "extension" probe) are designed so that they can anneal to each other only in the presence of the target, thus forming a structure called a three-way junction (3WJ) (Fig.…”

“…Following 3WJ formation, DNA polymerase extends the short extension probe so that a single-stranded promoter sequence on the template probe is converted into a functional double-stranded promoter for RNA polymerase, which in turn allows generation of multiple copies of an RNA signal. The RNA signal generated is further increased by additional rounds of extension and transcription (12,24). Thus, the SMART process is based on signal rather than target amplification.…”

“…Since the DNA and RNA polymerases function under the same conditions, the entire reaction takes place in a single tube. The RNA signal is detected and quantified by means of an enzyme-linked oligosorbent assay (ELOSA) in which color change is measured with a standard plate reader in a conventional 96-well microtiter plate format (12,24). The CytAMP MRSA assay detects the coa and mecA genes, thereby simultaneously identifying the presence of S. aureus and methicillin (oxacillin) resistance.…”

“…(C) Multiple copies of an RNA signal sequence are transcribed by T7 RNA polymerase from the template probe. This RNA sequence forms a substrate for additional rounds of extension and transcription in a second reaction that generates an unrelated signal sequence (12,24). All reactions occur concomitantly in a single microcentrifuge tube.…”

Fluorescence-based assay formats and signal amplification strategies for DNA microarray analysis

“…These assays rely on the fact that only the doublestranded promoter is fully functional, and allows T7 RNA polymerase to generate multiple copies of RNA molecules (Chan and Fox, 1999). Wharam et al (2001) have reported a dual enzyme technology, termed signal mediated amplification of RNA technology (SMART), which is based on the formation of a three-way junction (3WJ) structure (Fig. 8).…”