Back to results
US 2019/0292581 A1Published application
Proximity assays for detecting nucleic acids and proteins in a single cell
AssigneeFluidigm Corporation
InventorsCamila Egidio, Ramesh Ramakrishnan, David Ruff
Life of this patent
Estimating the term…
Abstract
Methods and reagents for detection and analysis of nucleic acids and proteins using proximity extension assays.
Claims6
A method of detecting a target analyte in a single cell, the method comprising:
- a) isolating the single cell;
- b) incubating the single cell in a lysing buffer comprising a detergent present at a concentration below the critical micelle concentration to obtain a cell lysate;
- c) incubating the cell lysate with two or more proximity extension probes in a binding reaction at an incubation temperature from about 15° C. to about 50° C. for a length of time from about 5 minutes to about 6 hours under conditions where the proximity extension probes bind to the target analyte, if present, in the cell lysate;
- d) incubating the binding reaction with an extension mix that comprises a polymerase, wherein hybridized oligonucleotide components of the proximity extension probe are extended by the polymerase to produce extension products;
- e) detecting the extension products.
2 more elements
A method of detecting a target analyte in a single cell, the method comprising:
- a) isolating the single cell, wherein isolating the single cell comprises isolation of individual cells into droplets in a microfluidic device;
- b) incubating the single cell in a lysing buffer comprising a detergent present at a concentration below the critical micelle concentration to obtain a cell lysate;
- c) incubating the cell lysate with two or more proximity extension probes in a binding reaction at an incubation temperature from about 15° C. to about 50° C. for a length of time from about 5 minutes to about 6 hours under conditions where the proximity extension probes bind to the target analyte, if present, in the cell lysate;
- d) incubating the binding reaction with an extension mix that comprises a polymerase, wherein hybridized oligonucleotide components of the proximity extension probe are extended by the polymerase to produce extension products;
- e) detecting the extension products.
2 more elements
A proximity extension detection probe set for detecting interaction of a protein with a single-stranded nucleic acid, wherein the probe set comprises a first proximity probe that comprises a binding region that binds to the protein and a first oligonucleotide comprising an interacting region;
- and a second proximity probe that comprises an oligonucleotide that comprises a segment that hybridizes to the single stranded nucleic acid and a segment that comprises an interacting region that is complementary to the interacting region of the first proximity probe, wherein, when the protein is bound to the single-stranded nucleic acid, the interacting region of the first probe hybridizes to the complementary segment of the second probe.
Dependencies are inferred from the claim text.