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 analyzing a biological sample, comprising:
(a) permeabilizing said biological sample, wherein said biological sample comprises a plurality of cells, wherein a cell of said plurality of cells comprises cellular nucleic acid molecules having a relative three-dimensional (3D) spatial orientation within said cell;
(b) generating a 3D matrix comprising said cellular nucleic acid molecules attached thereto, wherein said 3D matrix preserves said relative 3D spatial orientation;
(c) contacting said 3D matrix with reagents to selectively remove a non-nucleic acid component from said biological sample; and
(d) using 3D fluorescence imaging to identify said cellular nucleic acid molecules and said relative 3D spatial orientation of said cellular nucleic acid molecules within said cell.
2. The method of claim 1 , wherein (d) comprises hybridizing fluorescently labeled oligonucleotides to said cellular nucleic acid molecules or derivatives thereof and imaging said 3D matrix to detect said fluorescently labeled oligonucleotides.
3. The method of claim 2 , further comprising, (i) stripping said fluorescently labeled oligonucleotides from said cellular nucleic acid molecules or derivatives thereof; (ii) hybridizing additional fluorescently labeled oligonucleotides to said cellular nucleic acid molecules or derivatives thereof; and (iii) imaging said 3D matrix to detect said additional fluorescently labeled oligonucleotides.
4. The method of claim 2 , further comprising, prior to (d), contacting said cellular nucleic acid molecules with a plurality of nucleic acid probes and wherein (d) comprises hybridizing said fluorescently labeled oligonucleotides to said plurality of nucleic acid probes.
5. The method of claim 4 , further comprising, (i) stripping said fluorescently labeled oligonucleotides from said plurality of nucleic acid probes; (ii) hybridizing additional fluorescently labeled oligonucleotides to said plurality of nucleic acid probes; and (iii) imaging said 3D matrix to detect said additional fluorescently labeled oligonucleotides.
6. The method of claim 1 , further comprising, prior to (d), contacting said cellular nucleic acid molecules with a plurality of nucleic acid probes and wherein (d) comprises sequencing at least a portion of said plurality of nucleic acid probes.
7. The method of claim 6 , wherein said sequencing is sequencing-by-ligation.
8. The method of claim 6 , wherein said sequencing is sequencing-by-synthesis.
9. The method of claim 6 , wherein said sequencing is sequencing-by-hybridization.
10. The method of claim 1 , wherein (d) comprises sequencing said cellular nucleic acid molecules or derivatives thereof.
11. The method of claim 1 , wherein, prior to (b), said cellular nucleic acid molecules comprise a functional moiety and wherein said functional moiety is attached to said 3D matrix.
12. The method of claim 11 , wherein said functional moiety comprises acrydite, wherein (b) comprises contacting said biological sample with a matrix forming material and polymerizing said matrix forming material to generate said 3D matrix, and wherein said acrydite is polymerized into said 3D matrix thereby attaching said cellular nucleic acid molecules to said 3D matrix.
13. The method of claim 1 , wherein said cellular nucleic acid molecules are attached to said 3D matrix through a non-covalent interaction.
14. The method of claim 13 , wherein said non-covalent interaction is hydrogen bonding.
15. The method of claim 1 , wherein said cellular nucleic acid molecules are attached to said 3D matrix through a ligand-ligand binding pair, wherein a ligand of said ligand-ligand binding pair comprises a functional moiety that is attached to said 3D matrix.
16. The method of claim 15 , wherein said functional moiety comprises acrydite, wherein (b) comprises contacting said biological sample with a matrix forming material and polymerizing said matrix forming material to generate said 3D matrix, and wherein said acrydite is polymerized into said 3D matrix.
17. The method of claim 1 , wherein said reagents comprise a protease, an organic solvent or a denaturant.
18. The method of claim 1 , wherein said reagents comprise a detergent.
19. The method of claim 18 , wherein said reagents further comprise a protease.
20. The method of claim 1 , wherein said biological sample is a formalin-fixed, wax embedded tissue sample on a glass slide.
21. The method of claim 1 , further comprising, subsequent to (d), performing a 3D reconstruction of said cellular nucleic acid molecules within said cell.