Granted Patent B2

US 12,203,134 · App. 18/087,440 · Granted Jan 21, 2025

Inventors: Monica Nagendran (Pleasanton, CA); Jonathan Cheng (Pleasanton, CA); Eswar Prasad Ramachandran Iyer (Oakland, CA)

Assignee: 10x Genomics, Inc.

C12Q1/6841C12Q1/6806C12Q1/6844C12Q2600/16

Abstract

Claims (46)

1. A method of determining mislocalization of a nucleic acid within a tissue sample, the method comprising:

(a) obtaining a first image of the tissue sample, wherein the tissue sample is stained with a biomarker that is specific for a second analyte having colocalized expression with the nucleic acid;

(b) hybridizing the nucleic acid to a capture domain of a capture probe on an array, wherein the array comprises a plurality of capture probes, wherein the plurality of capture probes comprise a capture domain;

(c) extending the capture probe using the nucleic acid as a template, thereby generating an extended capture probe on the array;

(d) hybridizing a padlock probe or a snail probe to the extended capture probe on the array;

(e) circularizing the padlock probe or the snail probe on the array;

(f) amplifying the padlock probe or the snail probe on the array, thereby generating an amplified circularized padlock probe or an amplified circularized snail probe on the array;

(g) hybridizing a plurality of detection probes to the amplified circularized padlock probe or the amplified circularized snail probe on the array, wherein a detection probe from the plurality of detection probes comprises:

a nucleic acid sequence that is substantially complementary to a sequence of the padlock probe or the snail probe, and

a detectable label;

(h) obtaining a second image of the tissue sample and detecting a signal intensity from the plurality of detection probes in the second image; and

(i) determining mislocalization of the nucleic acid within the tissue sample by comparing detection of the biomarker in the first image to the signal intensity of the detectable label in the second image.

2. The method of claim 1 , wherein the padlock probe or the snail probe comprises:

(i) a first sequence that is substantially complementary to a first portion of the extended capture probe, or a complement thereof;

(ii) a backbone sequence; and

(iii) a second sequence that is substantially complementary to a second portion of the extended capture probe, or a complement thereof.

3. The method of claim 2 , wherein the first sequence and the second sequence are substantially complementary to adjacent sequences of the extended capture probe, or a complement thereof.

4. The method of claim 2 , wherein the first sequence and the second sequence are substantially complementary to sequences of the extended capture probe, or a complement thereof, that are about 2 to 50 nucleotides apart.

5. The method of claim 4 , wherein the circularizing the padlock probe or the snail probe comprises filling in a gap between the first sequence and the second sequence using a polymerase.

6. The method of claim 2 , wherein the backbone sequence comprises a backbone barcode sequence unique to the padlock probe or the snail probe.

7. The method of claim 1 , wherein the circularizing the padlock probe or the snail probe comprises ligating the padlock probe or the snail probe.

8. The method of claim 7 , wherein the ligating comprises enzymatic ligation or chemical ligation.

9. The method of claim 8 , wherein the enzymatic ligation utilizes a T4 DNA ligase.

10. The method of claim 1 , wherein the amplifying comprises rolling circle amplification.

11. The method of claim 10 , wherein the amplifying comprises:

hybridizing one or more primers to the padlock probe or the snail probe; and

amplifying the padlock probe or the snail probe with a polymerase.

12. The method of claim 11 , wherein the polymerase is a Phi29 DNA polymerase.

13. The method of claim 11 , wherein the one or more primers are substantially complementary to a portion of the padlock probe or the snail probe.

14. The method of claim 1 , wherein the detection probe is about 10 to about 60 nucleotides in length.

15. The method of claim 1 , wherein the detectable label comprises a fluorophore.

16. The method of claim 2 , wherein the detection probe is substantially complementary to (i) a portion of the first sequence; (ii) a portion of the second sequence; (iii) a portion of the backbone sequence; and/or (iv) a portion of the nucleic acid or a complement thereof.

17. The method of claim 1 , wherein the tissue sample is permeabilized using a permeabilization agent prior to step (b).

18. The method of claim 17 , wherein the permeabilization agent comprises proteinase K or pepsin.

19. The method of claim 1 , wherein the tissue sample comprises a formalin-fixed paraffin embedded sample.

20. The method of claim 1 , wherein the tissue sample comprises a fresh frozen sample.

21. The method of claim 1 , wherein the tissue sample comprises a tissue section.

22. The method of claim 1 , further comprising varying thickness of the tissue sample to determine an optimal tissue thickness to minimize nucleic acid mislocalization in the tissue sample.

23. The method of claim 1 , wherein the tissue sample is stained prior to obtaining the first image of the tissue sample.

24. The method of claim 23 , wherein the tissue sample is stained in (a) using 4′,6-diamidino-2-phenylindole (DAPI), immunofluorescence, immunohistochemistry, hematoxylin, or eosin.

25. The method of claim 1 , further comprising varying permeabilization time to determine an optimal permeabilization time for minimizing nucleic acid mislocalization in the tissue sample.

26. The method of claim 1 , further comprising varying permeabilization temperature to determine an optimal permeabilization temperature for minimizing nucleic acid mislocalization in the tissue sample.

27. The method of claim 1 , wherein determining the mislocalization of the nucleic acid comprises measuring signal intensity from the plurality of detection probes in the second image and comparing the signal intensity to detection of the biomarker in the first image of the tissue sample.

28. The method of claim 1 , wherein the capture domain of the capture probe comprises a poly(T) sequence.

29. The method of claim 1 , wherein the nucleic acid is mRNA.

30. The method of claim 1 , wherein the nucleic acid is an mRNA molecule and the second analyte is a protein translated from the nucleic acid.

Assignments (1)

Continuity (3)

References Cited (400)

Cited By (34)

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