Granted Patent B2

US 12,270,071 · App. 17/127,455 · Granted Apr 8, 2025

Inventors: Malte Kühnemund (Solna, SE); Toon Verheyen (Solna, SE)

Assignee: 10X GENOMICS, INC.

C12Q1/6816C12Q1/6876C12Q2525/307C12Q2531/125C12Q2563/107

Abstract

Claims (25)

1. A method of detecting a nucleic acid in a sample, wherein the sample is a permeabilized cell sample or a permeabilized tissue sample, comprising:

(a) contacting the sample with a circularizable probe that binds to the nucleic acid in the sample, producing a circularized probe by circularizing the circularizable probe bound to the nucleic acid, and generating a rolling circle amplification product (RCP) from the circularized probe, wherein the RCP comprises a marker sequence that identifies the nucleic acid;

(b) after step (a), contacting the sample with (i) an initiator comprising one or more parts and (ii) a plurality of linear oligo hybridization chain reaction (LO-HCR) monomers comprising a first species and a second species, wherein at least a fraction of the plurality of LO-HCR monomers are labelled with a detectable label,

wherein the initiator hybridizes directly or indirectly to the marker sequence in the RCP and comprises an output domain,

wherein the first species and the second species are linear, single-stranded nucleic acid molecules and do not comprise a hairpin structure or stem-loop structure,

wherein each of the first species and the second species comprise an input domain and an output domain,

wherein the input domain of the first species is complementary to the output domain of the initiator and the output domain of the second species, and the output domain of the first species is complementary to the input domain of the second species;

(c) generating a polymeric LO-HCR product comprising the detectable label using the initiator and the plurality of LO-HCR monomers, wherein the polymeric LO-HCR product comprises a plurality of the first species and a plurality of the second species; and

(d) detecting the polymeric LO-HCR product comprising the detectable label in the sample, thereby detecting the nucleic acid in the sample.

2. The method of claim 1 , wherein the plurality of LO-HCR monomers do not comprise a repeating sequence having more than about 5, about 10, or about 20 nucleotides in length.

3. The method of claim 1 , wherein the nucleic acid is a cellular nucleic acid molecule.

4. The method of claim 1 , wherein the nucleic acid is a genomic DNA, a cDNA generated by reverse transcripted of an mRNA prior to step (a), an mRNA, an miRNA, or an lncRNA.

5. The method of claim 1 , wherein the sample is the permeabilized tissue sample and fixed on a solid support, and the nucleic acid is detected in situ.

6. The method of claim 1 , wherein step (c) comprises:

(i) contacting the RCP with the initiator and forming a hybridized complex comprising the initiator and the RCP,

(ii) adding the first species to the hybridized complex,

(iii) adding the second species to the hybridized complex, and

(iv) repeating steps (ii) to (iii) for one or more times, wherein the first species hybridizes to the initiator and the second species in the polymeric LO-HCR product.

7. The method of claim 1 , wherein the method further comprises generating multiple sequential LO-HCR products by repeating steps (b) to (c) using identically or differentially labelled LO-HCR monomers.

8. The method of claim 1 , wherein the nucleic acid is an RNA molecule.

9. The method of claim 1 , wherein the nucleic acid is detected in the permeabilized cell sample or the permeabilized tissue sample, wherein the permeabilized cell sample or the tissue sample is fixed on a solid support.

10. The method of claim 1 , wherein step (b) comprises contacting the sample simultaneously with the first species and the second species.

11. The method of claim 1 , wherein step (b) comprises contacting the sample with the first species and subsequently contacting the sample with the second species.

12. The method of claim 1 , wherein the initiator is pre-bound to the first species before step (b).

13. The method of claim 1 , wherein step (b) comprises: (i) forming a complex comprising the initiator and the marker sequence in the RCP by contacting the sample with the initiator with the marker sequence in the RCP, (ii) washing the sample to remove the initiator not on the complex, and (iii) contacting the sample with the first species and second species after step (ii).

Assignments (2)

Priority Claims (1)

Continuity (1)

References Cited (323)