Method for generating a three-dimensional nucleic acid containing matrix

Patent metadata

Field	Value
Publication number	US-10138509-B2
Application number	US-201414774282-A
Country	US
Kind code	B2
Filing date	Feb 26, 2014
Priority date	Mar 12, 2013
Publication date	Nov 27, 2018
Grant date	Nov 27, 2018

Abstract

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

First claim

What is claimed is:

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 p

Assignees

• Harvard College

Inventors

• Church George M
• Lee Je-Hyuk
• Terry Richard C
• Daugharthy Evan R

Classifications

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title