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US 12,601,007 B2Granted
Multiplex labeling of molecules by sequential hybridization barcoding
AssigneeCalifornia Institute of Technology
InventorsLong Cai, Eric Lubeck, Timur Zhiyentayev, Ahmet Coskun, Ting-Fang He, Chang Ho Sohn, Sheel Shah
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Abstract
The present invention, among other things, provides technologies for detecting and/or quantifying nucleic acids in cells, tissues, organs or organisms. In some embodiments, through sequential barcoding, the present invention provides methods for high-throughput profiling of a large number of targets, such as transcripts and/or DNA loci.
Claims44
A method, comprising steps of:
- (a) performing a first contacting step that comprises contacting a cell comprising a plurality of target nucleic acids, the target nucleic acids comprising nucleic acids obtained by subjecting single stranded nucleic acids in the cell to amplification, with a first plurality of detectably labeled oligonucleotides, each of which targets a nucleic acid and is labeled with a detectable moiety, so that the first plurality comprises at least:
- (i) a first oligonucleotide targeting a first nucleic acid in the cell, the first oligonucleotide being labeled with a first detectable moiety;
- and
- (ii) a second oligonucleotide targeting a second nucleic acid in the cell, the second oligonucleotide being labeled with a second detectable moiety;
- (b) imaging the cell, after the first contacting step so that interaction by hybridizations of oligonucleotides of the first plurality with their target nucleic acids are detected;
- (c) performing a second contacting step that comprises contacting the cell, with a second plurality of detectably labeled oligonucleotides, which second plurality includes oligonucleotides targeting nucleic acids that are targeted by the first plurality so that the second plurality comprises at least:
- (i) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, targeting the first nucleic acid;
- and
- (ii) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide, targeting the second nucleic acid, wherein the second plurality differs from the first plurality in that at least one of the oligonucleotides present in the second plurality is labeled with a different detectable moiety than the corresponding oligonucleotide targeting the same nucleic acid in the first plurality, so that, in the second plurality:
- (iii) the third oligonucleotide is labeled with the first detectable moiety, the second detectable moiety or a third detectable moiety;
- and
- (iv) the fourth oligonucleotide is labeled with the first detectable moiety, the second detectable moiety, the third detectable moiety, or a fourth detectable moiety, wherein either the third oligonucleotide is labeled with a different detectable moiety than was the first oligonucleotide, or the fourth oligonucleotide is labeled with a different detectable moiety than was the second oligonucleotide, or both;
- (d) imaging the cell after the second contacting step so that interaction by hybridization of oligonucleotides of the second plurality with their target nucleic acids is detected;
- and
- (e) optionally repeating the contacting and imaging steps, each time with a new plurality of detectably labeled oligonucleotides comprising oligonucleotides that target nucleic acids targeted by the first and second pluralities, wherein each utilized plurality differs from each other utilized plurality, due to at least one difference in detectable moiety labeling of oligonucleotides targeting the same nucleic acid,
- so that the detected signals barcode the first target nucleic acid, and the second target nucleic acid in the cell, wherein the target nucleic acids can be differentiated by a difference in their barcodes.
13 more elements
A method, comprising steps of:
- (a) performing a first contacting step that comprises contacting a plurality of target nucleic acids, the target nucleic acids comprising transcripts or nucleic acids obtained by subjecting intermediate oligonucleotides to amplification, with a first plurality of detectably labeled oligonucleotides, each of which targets a nucleic acid and is labeled with a detectable moiety, so that the first plurality comprises at least:
- (i) a first oligonucleotide targeting a first nucleic acid, the first oligonucleotide being labeled with a first detectable moiety;
- and
- (ii) a second oligonucleotide targeting a second nucleic acid, the second oligonucleotide being labeled with a second detectable moiety;
- (b) imaging the target nucleic acids, after the first contacting step so that interaction by hybridizations of oligonucleotides of the first plurality with their target nucleic acids are detected;
- (c) performing a second contacting step that comprises contacting the target nucleic acids, with a second plurality of detectably labeled oligonucleotides, which second plurality includes oligonucleotides targeting nucleic acids that are targeted by the first plurality so that the second plurality comprises at least:
- (i) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, targeting the first nucleic acid;
- and
- (ii) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide, targeting the second nucleic acid, wherein the second plurality differs from the first plurality in that at least one of the oligonucleotides present in the second plurality is labeled with a different detectable moiety than the corresponding oligonucleotide targeting the same nucleic acid in the first plurality, so that, in the second plurality:
- (iii) the third oligonucleotide is labeled with the first detectable moiety, the second detectable moiety or a third detectable moiety;
- and
- (iv) the fourth oligonucleotide is labeled with the first detectable moiety, the second detectable moiety, the third detectable moiety, or a fourth detectable moiety, wherein either the third oligonucleotide is labeled with a different detectable moiety than was the first oligonucleotide, or the fourth oligonucleotide is labeled with a different detectable moiety than was the second oligonucleotide, or both;
- (d) imaging the nucleic acids after the second contacting step so that interaction by hybridization of oligonucleotides of the second plurality with their target nucleic acids is detected;
- and
- (e) optionally repeating the contacting and imaging steps, each time with a new plurality of detectably labeled oligonucleotides comprising oligonucleotides that target nucleic acids targeted by the first and second pluralities, wherein each utilized plurality differs from each other utilized plurality, due to at least one difference in detectable moiety labeling of oligonucleotides targeting the same nucleic acid,
- so that the detected signals barcode the first target nucleic acid, and the second target nucleic acid, wherein the target nucleic acids can be differentiated by a difference in their barcodes.
13 more elements
Dependencies are inferred from the claim text.