Method for generating a three-dimensional nucleic acid containing matrix
Methods of making a three-dimensional matrix of nucleic acids within a cell is provided.
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 performing isothermal enzymatic amplification on the plurality of nucleic acids.
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.
37. The method of claim 1 , wherein the plurality of nucleic acids are circular.
38. The method of claim 8 , 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.
40. The method of claim 7 , wherein each of the amplicons is coupled to the three-dimensional polymerized matrix upon formation.
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.
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.