Digital nuclease detection compositions and methods

Abstract

In certain embodiments, the present invention provides a detection composition comprising a picodroplet comprising (a) an aqueous solution, and (b) a substrate probe comprising (i) an oligonucleotide of 2 to 75 nucleotides in length, (ii) a fluorophore operably linked to the oligonucleotide, and (iii) a quencher operably linked to the oligonucleotide. As used herein, the term “picodroplet” comprises a liquid droplet that has a volume of 0.014 to 2.6 picoliters. In certain embodiments, the present invention provides a method of detecting at least one individual nuclease molecule present in a sample, comprising contacting an aqueous sample suspected of containing at least one nuclease with at least one detection composition comprising a picodroplet comprising (a) an aqueous solution, and (b) a substrate probe comprising (i) an oligonucleotide of 2 to 75 nucleotides in length, (ii) a fluorophore operably linked to the oligonucleotide, and (iii) a quencher operably linked to the oligonucleotide to form an aqueous reaction mixture; emulsifying the aqueous mixture in oil to form picoliter-scale droplets in an emulsion, (c) incubating the picoliter-scale droplets in the emulsion in order for the nuclease, if present, to digest the substrate probes linked to the microbeads; recovering the microbeads; and detecting fluorescence emitting from the microbeads.

Dependencies are inferred from the claim text.