Detection of viral RNAs at ambient temperature via reporter proteins produced through the target-splinted ligation of DNA probes.

Nucleic acid assays are not typically deployable in point-of-care settings because they require costly and sophisticated equipment for the control of the reaction temperature and for the detection of the signal. Here we report an instrument-free assay for the accurate and multiplexed detection of nucleic acids at ambient temperature. The assay, which we named INSPECTR (for internal splint-pairing expression-cassette translation reaction), leverages the target-specific splinted ligation of DNA probes to generate expression cassettes that can be flexibly designed for the cell-free synthesis of reporter proteins, with enzymatic reporters allowing for a linear detection range spanning four orders of magnitude and peptide reporters (which can be mapped to unique targets) enabling highly multiplexed visual detection.

Linear nicking endonuclease-mediated strand-displacement DNA amplification.

We describe a method for linear isothermal DNA amplification using nicking endonuclease-mediated strand displacement by a DNA polymerase. The nicking of one strand of a DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis. As the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated.

A device for automated hydrodynamic shearing of genomic DNA.

We describe a device for automated fragmentation of genomic DNA by hydrodynamic shearing using a filter screen with uniform pores. Human genomic DNA can be fragmented reproducibly to a range of 2000-12,000 bp by using various fluid flow rates and screens with 0.5-10-microm pores.