METHOD AND PRODUCT FOR LOCALIZED OR SPATIAL DETECTION OF NUCLEIC ACID IN A TISSUE SAMPLE

• US 20140066318A1
• Filed: 03/13/2012
• Published: 03/06/2014
• Est. Priority Date: 04/13/2011
• Status: Active Grant

• Current Assignee: 10X Genomics Sweden AB (10X Genomics Incorporated)
• Original Assignee: Spatial Transcriptomics AB (10X Genomics Incorporated)
• Inventors: Frisen, Jonas, Sthl, Patrik, Lundeberg, Joakim
• Granted Patent: US 10,030,261 B2
• Time in Patent Office: Days
• US Class Current: 506/3
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2543/101 in situ amplification
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Y 600/00 Ligases (6.)
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 1/42 Low-temperature sample treatment, e.g. cryofixation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 50/20 Heterogeneous data integration
• CPC Class Codes: G16B 50/30 Data warehousing; Computing architectures

Abstract

The present invention relates to methods and products for the localized or spatial detection of nucleic acid in a tissue sample and in particular to a method for localized detection of nucleic acid in a tissue sample comprising: (a) providing an array comprising a substrate on which multiple species of capture probes are directly or indirectly immobilized such that each species occupies a distinct position on the array and is oriented to have a free 3′ end to enable said probe to function as a primer for a primer extension or ligation reaction, wherein each species of said capture probe comprises a nucleic acid molecule with 5′ to 3′: (i) a positional domain that corresponds to the position of the capture probe on the array, and (ii) a capture domain; (b) contacting said array with a tissue sample such that the position of a capture probe on the array may be correlated with a position in the tissue sample and allowing nucleic acid of the tissue sample to hybridize to the capture domain in said capture probes; (c) generating DNA molecules from the captured nucleic acid molecules using said capture probes as extension or ligation primers, wherein said extended or ligated DNA molecules are tagged by virtue of the positional domain; (d) optionally generating a complementary strand of said tagged DNA and/or optionally amplifying said tagged DNA; (e) releasing at least part of the tagged DNA molecules and/or their complements or amplicons from the surface of the array, wherein said part includes the positional domain or a complement thereof; and (f) directly or indirectly analyzing the sequence of the released DNA molecules.

Claims

1. A method for localized detection of nucleic acid in a tissue sample comprising:

(a) providing an array comprising a substrate on which multiple species of capture probes are directly or indirectly immobilized such that each species occupies a distinct position on the array and is oriented to have a free 3′

end to enable said probe to function as a primer for a primer extension or ligation reaction, wherein each species of said capture probe comprises a nucleic acid molecule with 5′

to 3′

;

(i) a positional domain that corresponds to the position of the capture probe on the array, and

(ii) a capture domain;

(b) contacting said array with a tissue sample such that the position of a capture probe on the array may be correlated with a position in the tissue sample and allowing nucleic acid of the tissue sample to hybridize to the capture domain in said capture probes;

(c) generating DNA molecules from the captured nucleic acid molecules using said capture probes as extension or ligation primers, wherein said extended or ligated DNA molecules are tagged by virtue of the positional domain;

(d) optionally generating a complementary strand of said tagged DNA and/or optionally amplifying said tagged DNA;

(e) releasing at least part of the tagged DNA molecules and/or their complements or amplicons from the surface of the array, wherein said part includes the positional domain or a complement thereof; and

(f) directly or indirectly analyzing the sequence of the released DNA molecules.

2. The method of claim 1, further comprising step (g) correlating said sequence analysis information with an image of said tissue sample, wherein the tissue sample is imaged before or after step (c).

3. The method of claim 1 being a method for determining and/or analyzing a transcriptome of a tissue sample comprising:

(a) providing an array comprising a substrate on which multiple species of capture probes are directly or indirectly immobilized such that each species occupies a distinct position on the array and is oriented to have a free 3′

end to enable said probe to function as a reverse transcriptase (RT) primer, wherein each species of said capture probe comprises a nucleic acid molecule with 5′

to 3′

;

(i) a positional domain that corresponds to the position of the capture probe on the array; and

(ii) a capture domain;

(b) contacting said array with a tissue sample such that the position of a capture probe on the array may be correlated with a position in the tissue sample and allowing RNA of the tissue sample to hybridize to the capture domain in said capture probes;

(c) generating cDNA molecules from the captured RNA molecules using said capture probes as RT primers and optionally amplifying said cDNA molecules;

(d) releasing at least part of the cDNA molecules and optionally their amplicons from the surface of the array, wherein said released molecule may be a first strand and/or second strand cDNA molecule or an amplicon thereof and wherein said part includes the positional domain or a complement thereof;

(e) directly or indirectly analyzing the sequence of the released DNA molecules; and

optionally

(f) correlating said sequence analysis information with an image of said tissue sample, wherein the tissue sample is imaged before or after step (c).

4. The method according to claim 1, wherein the capture probes are DNA molecules.

5. The method according to claim 1, wherein the capture probes further comprise a universal domain which is 5′

relative to the positional domain, wherein said universal domain comprises;

(i) an amplification domain, for amplifying the generated DNA molecules; and

/or

(ii) a cleavage domain for releasing the generated DNA molecules from the surface of the array.

6. The method according to claim 1, wherein the positional domain of each species of capture probe comprises a unique barcode sequence.

7. The method according to claim 1, wherein the capture domain comprises a poly-T DNA oligonucleotide comprising at least 10 deoxythymidine residues and/or a random or degenerate oligonucleotide sequence.

8. The method according to claim 1, wherein the capture probes are directly immobilized on the array surface by their 5′

end.

9. The method according to claim 1, wherein the capture probes are indirectly immobilized on the array surface by hybridization to a surface probe, wherein the capture domain of the capture probes comprises an upstream sequence that is capable of hybridizing to 5′

end of surface probes that are immobilized on the array.

10. The method of claim 9, wherein the surface probes are immobilized to the array surface by their 3′

end.

11. The method according to claim 9, wherein the surface probes comprise a sequence that is complementary to:

(i) at least part of the capture domain;

(ii) the positional domain; and

(iii) at least part of the universal amplification domain.

12. The method according to claim 1, wherein said complementary strand or said second strand cDNA molecule is generated before or after said tagged DNA molecules or said cDNA molecules are released from the surface of the array.

13. The method according to claim 1, wherein a strand displacement polymerase is used for the synthesis of the complementary strand of the second strand of cDNA.

14. The method according to claim 1, wherein random primers are used for complementary strand or second strand cDNA synthesis.

15. The method of claim 14, wherein the random primers comprise an amplification domain.

16. The method of claim 15, wherein the amplification domain of the random primers consists of a nucleotide sequence that is different to the amplification domain of the capture probe.

17. The method according to claim 1, wherein adaptors are ligated to one or both ends of the tagged molecules or their complements or the cDNA molecules.

18. The method according to claim 1, further comprising the step of amplifying the tagged molecules or cDNA molecules prior to sequence analysis.

19. The method of claim 18, wherein the amplification step is performed after the release of the tagged DNA or cDNA molecules from the substrate of the array, or wherein the amplification step is performed in situ on the array.

20. The method according to claim 18, wherein the step of amplifying comprises a PCR.

21. The method according to claim 1, wherein the molecules are released from the surface of the array by:

(i) nucleic acid cleavage;

(ii) denaturation; and

/or

(iii) physical means.

22. The method of claim 21, wherein the molecules are released by enzymatic cleavage of a cleavage domain, which is located in the universal domain or positional domain of the capture probe.

23. The method of claim 21, wherein the molecules are released by denaturation and/or physical means.

24. The method of according to claim 1, further comprising a step of purifying the released molecules prior to sequencing.

25. The method according to claim 1, wherein the substrate is selected from the group consisting of glass, silicon, poly-L-lysine coated material, nitrocellulose, polystyrene, cyclic olefin copolymers (COCs), cyclic olefin polymers (COPs), polypropylene, polyethylene, and polycarbonate.

26. The method according to claim 1, wherein the tissue sample is a tissue section.

27. The method of claim 26, wherein the tissue section is prepared using a fixed tissue.

28. The method according to claim 1, further comprising a step of rehydrating the tissue sample after contacting the sample with the array and prior to step (c).

29. The method according to claim 1, wherein the tissue sample is from a plant, animal or fungus.

30. The method according to claim 1, further comprising a step of washing the array to remove residual tissue, wherein said step is prior to the release of the molecules from the array.

31. The method according to claim 1, wherein the array comprises at least one positional marker to enable orientation of the tissue sample on the array.

32. The method of claim 31, wherein the array comprises at least 10, 50, 100, 200, 500, 1000 or 2000 positional markers in distinct positions on the surface of the array.

33. The method according to claim 31, wherein the positional markers are capable of hybridizing to a labeled marker nucleic acid molecule.

34. The method according to claim 2, wherein the tissue sample is imaged using light, bright field, dark field, phase contrast, fluorescence, reflection, interference or confocal microscopy or a combination thereof.

35. The method of claim 34, wherein the tissue sample is imaged using fluorescence microscopy.

36. The method according to claim 1, wherein the sequence analysis step includes a step of sequencing.

42. The method according to claim 1 being a method for localized detection of DNA in a tissue sample comprising:

(a) providing an array comprising a substrate on which multiple species of capture probes are directly or indirectly immobilized such that each species occupies a distinct position on the array and is oriented to have a free 3′

end to enable said probe to function as a primer for a primer extension or ligation reaction, wherein each species of said capture probe comprises a nucleic acid molecule with 5′

to 3′

;

(i) a positional domain that corresponds to the position of the capture probe on the array, and

(ii) a capture domain;

(b) contacting said array with a tissue sample such that the position of a capture probe on the array may be correlated with a position in the tissue sample and allowing DNA of the tissue sample to hybridize to the capture domain in said capture probes;

(c) fragmenting DNA in said tissue sample, wherein said fragmentation is carried out before, during or after contacting the array with the tissue sample in step (b);

(d) extending said capture probes in a primer extension reaction using the captured DNA fragments as templates to generate extended DNA molecules, or ligating the captured DNA fragments to the capture probes in a ligation reaction to generate ligated DNA molecules, wherein said extended or ligated DNA molecules are tagged by virtue of the positional domain;

(e) optionally generating a complementary strand of said tagged DNA and/or optionally amplifying said tagged DNA;

(f) releasing at least part of the tagged DNA molecules and/or their complements and/or amplicons from the surface of the array, wherein said part includes the positional domain or a complement thereof;

(g) directly or indirectly analyzing the sequence of the released DNA molecules; and

optionally;

(h) correlating said sequence analysis information with an image of said tissue sample, wherein the tissue sample is imaged before or after step (d).

43. The method of claim 42 comprising:

(a) providing an array comprising a substrate on which multiple species of capture probes are directly or indirectly immobilized such that each species occupies a distinct position on the array and is oriented to have a free 3′

end to enable said probe to function as a primer for a primer extension or ligation reaction, wherein each species of said capture probe comprises a nucleic acid molecule with 5′

to 3′

;

(i) a positional domain that corresponds to the position of the capture probe on the array, and

(ii) a capture domain;

(b) contacting said array with a tissue sample such that the position of a capture probe on the array may be correlated with a position in the tissue sample;

(c) fragmenting DNA in said tissue sample, wherein said fragmentation is carried out before, during or after contacting the array with the tissue sample in step (b);

(d) providing said DNA fragments with a binding domain which is capable of hybridizing to said capture domain;

(e) allowing said DNA fragments to hybridize to the capture domain in said capture probes;

(f) extending said capture probes in a primer extension reaction using the captured DNA fragments as templates to generate extended DNA molecules, or ligating the captured DNA fragments to the capture probes in a ligation reaction to generate ligated DNA molecules, wherein said extended or ligated DNA molecules are tagged by virtue of the positional domain;

(g) optionally generating a complementary strand of said tagged DNA and/or optionally amplifying the tagged DNA

(h) releasing at least part of the tagged DNA molecules and/or their complements and/or amplicons from the surface of the array, wherein said part includes the positional domain or a complement thereof;

(i) directly or indirectly analyzing the sequence of the released DNA molecules; and

optionally

(j) correlating said sequence analysis information with an image of said tissue sample, wherein the tissue sample is imaged before or after step (f).

44. The method of claim 21, wherein the molecules are released by applying hot water or buffer to the array.

45. The method of claim 27, wherein the fixed tissue is a formalin-fixed paraffin-embedded (FFPE) tissue or deep-frozen tissue.

46. The method according to claim 33, wherein the marker nucleic acid molecule is fluorescently labeled.

47. The method of claim 36, wherein the sequencing step comprises a sequencing reaction based on reversible dye-terminators.

37. A method for making or producing an array (i) configured to capture nucleic acid from a tissue sample that is contacted with said array;

or (ii) configured localized detection of nucleic acid in a tissue sample, said method comprising immobilizing, directly or indirectly, multiple species of capture probe to an array substrate, wherein each species of said capture probe comprises a nucleic acid molecule with 5′

to 3′

;

(i) a positional domain that corresponds to the position of the capture probe on the array; and

(ii) a capture domain.

38. The method of claim 37, wherein each species of capture probe occupies a distinct position on the array.

39. The method according to claim 37, further comprising any one or more of the following steps:

(a) immobilizing directly or indirectly multiple surface probes to an array substrate, wherein the surface probes comprise;

(i) a domain capable of hybridizing to part of the capture domain oligonucleotide (a part not involved in capturing the nucleic acid);

(ii) a complementary positional domain; and

(iii) a complementary universal domain;

(b) hybridizing to the surface probes immobilized on the array capture domain oligonucleotides and universal domain oligonucleotides;

(c) extending the universal domain oligonucleotides, by templated polymerization, to generate the positional domain of the capture probe; and

(d) ligating the positional domain to the capture domain oligonucleotide to produce the capture oligonucleotide.

41. The method according to claim 37, wherein:

(a) the surface probes are;

(i) immobilized to the array surface by their 3′

end; and

/or

(ii) comprise a sequence that is complementary to;

(1) at least part of the capture domain;

(2) the positional domain; and

(3) at least part of the universal amplification domain;

(b) the substrate is selected from the group consisting of glass, silicon, poly-L-lysine coated material, nitrocellulose, polystyrene, cyclic olefin copolymers (COCs), cyclic olefin polymers (COPs), polypropylene, polyethylene, and polycarbonate;

(c) the tissue sample is a tissue section, optionally prepared using a fixed tissue;

(d) the array comprises at least one positional marker to enable orientation of the tissue sample on the array; and

(e) the positional markers are capable of hybridizing to a labeled marker nucleic acid molecule.

40. An array configured to:

(a) capture nucleic acid from a tissue sample that is contacted with said array;

or

(b) carry out localized detection of nucleic acid in a tissue sample, comprising a substrate on which multiple species of capture probes are directly or indirectly immobilized such that each species occupies a distinct position on the array and is oriented to have a free 3′

end to enable said probe to function as an extension or ligation primer, wherein each species of said capture probe comprises a nucleic acid molecule with 5′

to 3′

;

(i) a positional domain that corresponds to the position of the capture probe on the array, and

(ii) a capture domain to capture nucleic acid of a tissue sample that is contacted with said array.

48. The array of claim 40, wherein:

(a) the surface probes are;

(i) immobilized to the array surface by their 3′

end; and

/or

(ii) comprise a sequence that is complementary to;

(1) at least part of the capture domain;

(2) the positional domain; and

(3) at least part of the universal amplification domain;

(b) the substrate is selected from the group consisting of glass, silicon, poly-L-lysine coated material, nitrocellulose, polystyrene, cyclic olefin copolymers (COCs), cyclic olefin polymers (COPs), polypropylene, polyethylene, and polycarbonate;

(c) the tissue sample is a tissue section, optionally prepared using a fixed tissue;

(d) the array comprises at least one positional marker to enable orientation of the tissue sample on the array; and

(e) the positional markers are capable of hybridizing to a labeled marker nucleic acid molecule.