Highly-multiplexed fluorescent imaging
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsGarry P. Nolan, Nikolay Samusik, Julia Kennedy-Darling, Yury Goltsev
Life of this patent
Estimating the term…
Abstract
Provided herein is a method and system for analyzing a sample. In some embodiments the method makes use of a plurality of capture agents that are each linked to a different oligonucleotide and a corresponding plurality of labeled nucleic acid probes, wherein each of the labeled nucleic acid probes specifically hybridizes with only one of the oligonucleotides. The sample is labeled with the capture agents en masse, and sub-sets of the capture agents are detected using iterative cycles using corresponding subsets of the labeled nucleic acid probes.
Claims21
A method for analyzing a sample comprising fixed cells, comprising the following steps, performed in order:
- (a) obtaining:
- i. a plurality of capture agents that are each linked to a different oligonucleotide;
- and
- ii. a corresponding plurality of labeled nucleic acid probes, wherein each of the labeled nucleic acid probes specifically hybridizes with only one of the oligonucleotides of (a)(i);
- (b) labeling the sample comprising fixed cells with the plurality of capture agents of (a)(i);
- (c) specifically hybridizing a first sub-set of the labeled nucleic acid probes of (a)(ii) with the sample comprising fixed cells, wherein the probes in the first sub-set are distinguishably labeled, to produce labeled probe/oligonucleotide duplexes;
- (d) reading the sample comprising fixed cells to obtain an image showing binding pattern for each of the probes hybridized in step (c);
- (e) completely removing the probes hybridized in step (c) from the sample comprising fixed cells by denaturation by incubating the sample in 70%-90% of a chemical denaturant in the absence of heating, wherein the chemical denaturant is DMSO or formamide and the denaturation is not by displacing the DNA probes using displacement probes, leaving the plurality of capture agents of (b) and their associated oligonucleotides still bound to the sample comprising fixed cells;
- and
- (f) repeating steps (c) and (d) multiple times with a different sub-set of the labeled nucleic acid probes of (a)(ii), each repeat followed by step (e) except for the final repeat, to produce a plurality of images of the sample comprising fixed cells, each image corresponding to a different sub-set of labeled nucleic acid probes used in (c).
Dependencies are inferred from the claim text.