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US 10,457,980 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.
Claims21
A method of providing a unique molecular barcode for each unique nucleic acid molecule in a plurality of nucleic acid molecules in a sample, comprising the steps of:
- (a) performing a first contacting step that involves contacting the sample with a first plurality of detectably labeled oligonucleotides, each of which targets a nucleic acid molecule and is labeled with a detectable moiety, so that the first plurality comprises at least:
- (i) a first oligonucleotide targeting a first nucleic acid molecule and labeled with a first detectable moiety;
- and
- (ii) a second oligonucleotide targeting a second nucleic acid molecule and labeled with a second detectable moiety;
- (b) imaging the sample after the first contacting step so that interaction by oligonucleotides of the first plurality with their target sequences is detected;
- (c) performing a second contacting step that involves contacting the sample with a second plurality of detectably labeled oligonucleotides, which second plurality includes oligonucleotides targeting nucleic acid molecules 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 molecule;
- and
- (ii) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide, targeting the second nucleic acid molecule,
- 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 molecule 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 sample after the second contacting step so that interaction by oligonucleotides of the second plurality with their target nucleic acid molecules 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-acid molecules 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 molecule,
- wherein steps (a) to (e) produce a unique molecular barcode for each unique nucleic acid molecule in the sample.
15 more elements
A method of providing a unique molecular barcode for each unique transcript or DNA molecule in a plurality of transcripts and DNA molecules in a sample, comprising the steps of:
- (a) performing a first contacting step that involves contacting the sample with a first plurality of detectably labeled oligonucleotides, each of which targets a transcript or DNA locus and is labeled with a detectable moiety, so that the first plurality comprises at least:
- (i) a first oligonucleotide targeting a first transcript or DNA locus and labeled with a first detectable moiety;
- and
- (ii) a second oligonucleotide targeting a second transcript or DNA locus and labeled with a second detectable moiety;
- (b) imaging the sample after the first contacting step so that hybridization by oligonucleotides of the first plurality with their target sequences is detected;
- (c) performing a second contacting step that involves contacting the sample with a second plurality of detectably labeled oligonucleotides, which second plurality includes oligonucleotides targeting transcripts and/or DNA loci 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 transcript or DNA locus;
- and
- (ii) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide, targeting the second transcript or DNA locus,
- 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 transcript or DNA locus 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 sample after the second contacting step so that hybridization by oligonucleotides of the second plurality with their target sequences 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 transcripts or DNA loci 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 transcript or DNA locus,
- wherein steps (a) to (e) produce a unique molecular barcode for each unique transcript or DNA molecule in the sample.
15 more elements
Dependencies are inferred from the claim text.