Method for generating a three-dimensional nucleic acid containing matrix

• US 10,138,509 B2
• Filed: 02/26/2014
• Issued: 11/27/2018
• Est. Priority Date: 03/12/2013
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Church, George M., Lee, Je-Hyuk, Terry, Richard C., Daugharthy, Evan R.
• Primary Examiner(s): Thomas, David C
• Application Number:
  US14/774,282
• Publication Number:
  US 20160024555A1
• Time in Patent Office: 1,735 Days
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• 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 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

Methods of making a three-dimensional matrix of nucleic acids within a cell is provided.

Claims

1. A method of identifying nucleic acids within a cell, comprising:

contacting a plurality of nucleic acids having a relative three-dimensional spatial relationship within the cell with a matrix-forming material in a manner to substantially retain the relative three-dimensional spatial relationship;

using the matrix-forming material to form a three-dimensional polymerized matrix including the nucleic acids of the plurality of nucleic acids covalently bound to the three-dimensional polymerized matrix; and

detecting signals from the nucleic acids or derivatives thereof, thereby identifying the nucleic acids.

2. The method of claim 1 further including the step of amplifying the nucleic acids to produce amplicons within the three-dimensional polymerized matrix.

3. The method of claim 2 further including the step of covalently bonding the amplicons to the three-dimensional polymerized matrix.

4. The method of claim 1 wherein the plurality of nucleic acids are contained within a biological sample and the matrix-forming material is introduced into the biological sample.

5. The method of claim 1 wherein the plurality of nucleic acids are contained within a cell and the matrix-forming material is introduced into the cell.

6. The method of claim 1 wherein the plurality of nucleic acids are contained within a tissue sample and the matrix-forming material is introduced into the tissue sample.

16. The method of claim 1 wherein the relative three-dimensional spatial relationship of each nucleic acid of the plurality of nucleic acids is immobilized within the three-dimensional polymerized matrix.

17. The method of claim 1 wherein the matrix-forming material comprises polyacrylamide, cellulose, alginate, polyamide, cross-linked agarose, cross-linked dextran or cross-linked polyethylene glycol.

24. The method of claim 1, wherein the three-dimensional polymerized matrix is formed by subjecting the matrix-forming material to cross-linking.

25. The method of claim 1, wherein the three-dimensional polymerized matrix is formed by subjecting the matrix-forming material to polymerization.

30. The method of claim 2, wherein amplifying the nucleic acids comprises performing polymerase chain reaction (PCR) on the nucleic acids.

31. The method of claim 2, wherein amplifying the nucleic acids comprises performing isothermal enzymatic amplification on the nucleic acids.

32. The method of claim 2, wherein the amplicons are labeled with a detectable label.

37. The method of claim 1, wherein the plurality of nucleic acids are circular.

39. The method of claim 1, wherein the matrix-forming material is used to form the three-dimensional polymerized matrix subsequent to contacting the plurality of nucleic acids with the matrix-forming material.

42. The method of claim 1, wherein detecting the signals comprises contacting a nucleic acid of the nucleic acids or derivatives thereof with a detectable label and detecting a signal from the detectable label.

43. The method of claim 1, wherein detecting the signals comprises contacting a nucleic acid of the nucleic acids or derivatives thereof with a plurality of detectable labels and detecting signals from the plurality of detectable labels.

7. A method of identifying one or more nucleic acids within a cell comprising:

contacting a plurality of nucleic acids having a relative three-dimensional spatial relationship within the cell with a matrix-forming material to form a three-dimensional polymerized matrix comprising the plurality of nucleic acids coupled to the three-dimensional polymerized matrix, wherein the relative three-dimensional spatial relationship is substantially retained in the three-dimensional polymerized matrix;

amplifying the plurality of nucleic acids to produce amplicons within the three-dimensional polymerized matrix;

coupling the amplicons to the three-dimensional polymerized matrix; and

detecting the amplicons to identify a sequence of the one or more nucleic acids.

9. The method of claim 7 wherein the plurality of nucleic acids are circular.

10. The method of claim 7 wherein the plurality of nucleic acids are contained within a biological sample and the matrix-forming material is introduced into the biological sample.

11. The method of claim 7 wherein the plurality of nucleic acids are contained within a cell and the matrix-forming material is introduced into the cell.

12. The method of claim 7 wherein the plurality of nucleic acids are contained within a tissue sample and the matrix-forming material is introduced into the tissue sample.

18. The method of claim 7 wherein the relative three-dimensional spatial relationship of each nucleic acid of the plurality of nucleic acids is immobilized within the three-dimensional polymerized matrix.

20. The method of claim 7 wherein contacting the plurality of nucleic acids having the relative three-dimensional spatial relationship within the cell with the matrix-forming material comprises subjecting the matrix-forming material to a polymerization inducing catalyst, ultraviolet (UV) light, or functional cross-linkers.

21. The method of claim 7 wherein coupling the amplicons to the three-dimensional polymerized matrix comprises covalently bonding the amplicons to the three-dimensional polymerized matrix.

22. The method of claim 7 wherein the three-dimensional polymerized matrix comprises the plurality of nucleic acids covalently bound to the three-dimensional polymerized matrix.

23. The method of claim 7 wherein the amplicons are labeled with a detectable label.

26. The method of claim 7, wherein the three-dimensional polymerized matrix is formed by subjecting the matrix-forming material to cross-linking.

27. The method of claim 7, wherein the three-dimensional polymerized matrix is formed by subjecting the matrix-forming material to polymerization.

33. The method of claim 7, wherein amplifying the plurality of nucleic acids comprises performing PCR on the plurality of nucleic acids.

34. The method of claim 7, wherein amplifying the plurality of nucleic acids comprises performing isothermal enzymatic amplification on the plurality of nucleic acids.

40. The method of claim 7, wherein each of the amplicons is coupled to the three-dimensional polymerized matrix upon formation.

8. A method of identifying a relative three-dimensional spatial relationship of one or more nucleic acids within a cell comprising:

contacting a plurality of nucleic acids having the relative three-dimensional spatial relationship within the cell with a matrix-forming material in a manner to substantially retain the relative three-dimensional spatial relationship;

using the matrix-forming material to form a three-dimensional polymerized matrix including the plurality of nucleic acids covalently bound to the three-dimensional polymerized matrix;

amplifying the plurality of nucleic acids to produce amplicons within the matrix, covalently bonding the amplicons to the three-dimensional polymerized matrix;

labeling the amplicons with a detectable label; and

imaging the amplicons to identify the relative three-dimensional spatial relationship of the one or more nucleic acids within the cell.

13. The method of claim 8 wherein the plurality of nucleic acids are contained within a biological sample and the matrix-forming material is introduced into the biological sample.

14. The method of claim 8 wherein the plurality of nucleic acids are contained within a cell and the matrix-forming material is introduced into the cell.

15. The method of claim 8 wherein the plurality of nucleic acids are contained within a tissue sample and the matrix-forming material is introduced into the tissue sample.

19. The method of claim 8 wherein the relative three-dimensional spatial relationship of each nucleic acid of the plurality of nucleic acids is immobilized within the three-dimensional polymerized matrix.

28. The method of claim 8, wherein the three-dimensional polymerized matrix is formed by subjecting the matrix-forming material to cross-linking.

29. The method of claim 8, wherein the three-dimensional polymerized matrix is formed by subjecting the matrix-forming material to polymerization.

35. The method of claim 8, wherein amplifying the plurality of nucleic acids comprises performing PCR on said plurality of nucleic acids.

36. The method of claim 8, wherein amplifying the plurality of nucleic acids comprises performing isothermal enzymatic amplification on the plurality of nucleic acids.

38. The method of claim 8, wherein the plurality of nucleic acids are circular.

41. The method of claim 8, wherein the matrix-forming material is used to form the three-dimensional polymerized matrix subsequent to contacting the plurality of nucleic acids with the matrix-forming material.