Methods for attaching cellular constituents to a matrix
The present invention relates to a method of covalently attaching a chromosome within a cell to a matrix including modifying a plurality of nucleotides within the chromosome to include a matrix attachment moiety wherein the chromosome contacts the matrix, and attaching the matrix attachment moiety of the plurality of nucleotides to the matrix, thereby attaching the chromosome to the matrix.
1. A method of covalently attaching a chromosome within a cell to a matrix comprising
modifying a plurality of nucleotides within the chromosome to include a matrix attachment moiety wherein the chromosome contacts the matrix, and
attaching the matrix attachment moiety of the plurality of nucleotides to the matrix, thereby attaching the chromosome to the matrix.
2. The method of claim 1 wherein a matrix-forming material is introduced into the cell and forms the matrix contacting the chromosome.
3. The method of claim 2 wherein the matrix-forming material is polymerized to form the matrix.
4. The method of claim 2 wherein the matrix-forming material is crosslinked to form the matrix.
5. The method of claim 2 wherein the matrix is one or more of polyacrylamide, cellulose, alginate, polyamide, crosslinked agarose, cross-linked dextran, or cross-linked polyethylene glycol.
6. The method of claim 1 wherein the matrix is expanded and additional nucleotides within the chromosome are modified to include a matrix attachment moiety and the matrix attachment moiety is attached to the matrix.
7. The method of claim 1 wherein the matrix is expanded one or more times.
8. The method of claim 1 wherein the matrix is repeatedly expanded.
9. The method of claim 1 wherein the matrix is expanded one or more times, and with each expansion, additional nucleotides within the chromosome are modified to include a matrix attachment moiety and the matrix attachment moiety is attached to the matrix.
10. The method of claim 1 wherein the matrix is repeatedly expanded, and with each expansion, additional nucleotides within the chromosome are modified to include a matrix attachment moiety and the matrix attachment moiety is attached to the matrix.
11. The method of claim 1 wherein the plurality of nucleotides are chemically modified to include the matrix attachment moiety.
12. The method of claim 1 wherein the plurality of nucleotides are chemically modified to include a binding site for a matrix attachment moiety directly or through a linker, and the matrix attachment moiety or the linker is bound to the binding site.
13. The method of claim 1 wherein the plurality of nucleotides are modified by being cleaved from an adjacent nucleotide to produce one or more free end terminal nucleotides, and
wherein a nucleotide including a matrix attachment moiety is connected directly or as part of an oligonucleotide to the one or more free end terminal nucleotides, and
the matrix attachment moiety is attached to the matrix.
14. The method of claim 13 wherein the one or more free end terminal nucleotides are processed to allow attachment of the nucleotide including a matrix attachment moiety or the oligonucleotide including the nucleotide including a matrix attachment moiety.
15. The method of claim 13 wherein the one or more free end terminal nucleotides are processed to create blunt ends to allow attachment of the nucleotide including a matrix attachment moiety or the oligonucleotide including the nucleotide including a matrix attachment moiety.
16. The method of claim 13 wherein the one or more free end terminal nucleotides are processed to create overhangs to allow attachment of the nucleotide including a matrix attachment moiety or the oligonucleotide including the nucleotide including a matrix attachment moiety.
17. The method of claim 13 wherein the chromosome is cleaved by a restriction enzyme, a DNase, a single strand cutter, a double strand cutter, or irradiation to create a plurality single stranded or double stranded breaks or gaps.
18. The method of claim 13 wherein the nucleotide including a matrix attachment moiety is ligated directly or via an oligonucleotide to the one or more free end terminal nucleotides.
19. The method of claim 13 wherein the nucleotide including a matrix attachment moiety is extended from the one or more free end terminal nucleotides.
20. The method of claim 13 wherein one or more modified nucleotides are added to a terminal nucleotide using a polymerase,
attaching a matrix attachment moiety to the one or more modified nucleotides, and
attaching the matrix attachment moiety to the matrix.
21. The method of claim 13 wherein one or more nucleotides are added to a terminal nucleotide using a polymerase,
wherein the one or more nucleotides are chemically modified to include a binding site for a matrix attachment moiety directly or through a linker, and
the matrix attachment moiety or the linker is bound to the binding site, and
the matrix attachment moiety is attached to the matrix.
22. The method of claim 1 wherein the matrix attachment moiety is a member selected from the group consisting of amine, amine reactive groups, acrydite, an acrydite modified entity, alkyne, biotin, azide, thiol, a thiol-modified entity, and entities suitable for click chemistry techniques.
23. The method of claim 1 wherein the matrix material is expanded in three dimensions creating breaks in chromosomal DNA producing one or more additional free end terminal nucleotides.
24. The method of claim 23 wherein a nucleotide including a matrix attachment moiety is connected directly or indirectly to the one or more additional free end terminal nucleotides, and
the matrix attachment moiety is attached to the matrix.
25. The method of claim 24 wherein the matrix attachment moiety is a member selected from the group consisting of amine, amine reactive groups, acrydite, an acrydite modified entity, alkyne, biotin, azide, thiol, a thiol-modified entity, and entities suitable for click chemistry techniques.
26. The method of claim 23 wherein the one or more additional free end terminal nucleotides are processed to allow attachment of a nucleotide including a matrix attachment moiety or an oligonucleotide including the nucleotide including a matrix attachment moiety.
27. The method of claim 23 wherein the one or more additional free end terminal nucleotides are processed to create blunt ends to allow attachment of a nucleotide including a matrix attachment moiety or an oligonucleotide including the nucleotide including a matrix attachment moiety.
28. The method of claim 23 wherein the one or more additional free end terminal nucleotides are processed to create overhangs to allow attachment of a nucleotide including a matrix attachment moiety or an oligonucleotide including the nucleotide including a matrix attachment moiety.
29. The method of claim 23 wherein a nucleotide including a matrix attachment moiety is ligated directly or via an oligonucleotide to the one or more additional free end terminal nucleotides.
30. The method of claim 23 wherein a nucleotide including a matrix attachment moiety is extended from the one or more additional free end terminal nucleotides.
31. The method of claim 23 wherein one or more modified nucleotides are added to the one or more additional free end terminal nucleotides using a polymerase,
attaching a plurality of matrix attachment moieties to the one or more modified nucleotides, and
attaching the matrix attachment moieties to the matrix.
32. The method of claim 23 wherein one or more nucleotides are added to the one or more additional free end terminal nucleotides using a polymerase,
wherein the one or more nucleotides are chemically modified to include a binding site for a matrix attachment moiety directly or through a linker, and
the matrix attachment moiety or the linker is bound to the binding site, and
the matrix attachment moiety is attached to the matrix.