Methods for Attaching Cellular Constituents to a Matrix

Abstract

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 - 41 . (canceled) 
     
     
         42 . A method of modeling structure of a chromosome in a cell comprising:
 (a) hybridizing to the chromosome a plurality of oligonucleotides,
 wherein each oligonucleotide comprises 
 (1) a hybridization region complementary to a sequence on the chromosome; and 
 (2) at least one region that is not complementary to the sequence on the chromosome; 
   (b) contacting the chromosome and the plurality of oligonucleotides hybridized thereto with a matrix-forming material;   (c) attaching the plurality of oligonucleotides to the matrix;   wherein the plurality of oligonucleotides attached to the matrix represents the structure of the chromosome in the cell;
 (d) treating the matrix with a protease and/or passivating the matrix; and; 
   (e) detecting the modeled chromosome structure.   
     
     
         43 . The method of  claim 42 , wherein each of the plurality of oligonucleotides comprise one or more detectable labels, and wherein the step of detecting the modeled chromosome structure comprises detecting the detectable label. 
     
     
         44 . The method of  claim 43 , wherein the detectable label comprises a fluorescent label, and wherein the step of detecting the detectable label comprises one or more of fluorescence microscopy, super-resolution microscopy, Stimulated Emission Depletion Microscopy (STED), structured Illumination Microscopy (SIM), and single-molecule super-resolution microscopy technologies selected from Photo-Activated Localization Microscopy (PALM), stochastic Optical Reconstruction Microscopy (STORM) and DNA-based Point Accumulation In Nanoscale Topography (DNA-PAINT). 
     
     
         45 . The method of  claim 43 , wherein prior to said detecting the detectable label, the matrix is expanded. 
     
     
         46 . The method of  claim 42 , wherein the step of detecting the modeled chromosome structure comprises hybridizing a secondary oligonucleotide comprising a detectable label to at least one of the non-complementary regions of each of the plurality of oligonucleotides, and detecting the detectable label. 
     
     
         47 . The method of  claim 46 , wherein the detectable label comprises a fluorescent label, and the step of detecting the detectable label comprises one or more of fluorescence microscopy, super-resolution microscopy, Stimulated Emission Depletion Microscopy (STED), structured Illumination Microscopy (SIM), and single-molecule super-resolution microscopy technologies selected from Photo-Activated Localization Microscopy (PALM), stochastic Optical Reconstruction Microscopy (STORM) and DNA-based Point Accumulation In Nanoscale Topography (DNA-PAINT). 
     
     
         48 . The method of  claim 46 , wherein prior to the step of detecting the detectable label, the matrix is expanded. 
     
     
         49 . The method of  claim 42 , wherein the step of detecting the modeled chromosome structure comprises amplification of at least a portion of each oligonucleotide of the plurality of oligonucleotides to produce an amplified product, wherein the amplified product comprises one or more detectable labels; and detecting the detectable label. 
     
     
         50 . The method of  claim 49 , wherein the amplification comprises rolling circle amplification. 
     
     
         51 . The method of  claim 49 , wherein the detectable label comprises a fluorescent label, and the step of detecting the detectable label comprises one or more of fluorescence microscopy, super-resolution microscopy, Stimulated Emission Depletion Microscopy (STED), structured Illumination Microscopy (SIM), and single-molecule super-resolution microscopy technologies selected from Photo-Activated Localization Microscopy (PALM), stochastic Optical Reconstruction Microscopy (STORM) and DNA-based Point Accumulation In Nanoscale Topography (DNA-PAINT). 
     
     
         52 . The method of  claim 49 , wherein prior to the step of detecting the detectable label, the matrix is expanded. 
     
     
         53 . The method of  claim 42 , wherein the step of detecting the modeled chromosome structure comprises sequencing at least a portion of each oligonucleotide of the plurality of oligonucleotides, wherein sequencing comprises the use of one or more nucleotides comprising a detectable label, and detecting the detectable label. 
     
     
         54 . The method of  claim 53 , wherein the detectable label comprises a fluorescent label, and the step of detecting the detectable label comprises one or more of fluorescence microscopy, super-resolution microscopy, Stimulated Emission Depletion Microscopy (STED), structured Illumination Microscopy (SIM), and single-molecule super-resolution microscopy technologies selected from Photo-Activated Localization Microscopy (PALM), stochastic Optical Reconstruction Microscopy (STORM) and DNA-based Point Accumulation In Nanoscale Topography (DNA-PAINT). 
     
     
         55 . The method of  claim 53 , wherein prior to the step of detecting the detectable label, the matrix is expanded. 
     
     
         56 . The method of  claim 42 , wherein the matrix is expanded and additional oligonucleotides are hybridized to the chromosome and attached to the matrix. 
     
     
         57 . The method of  claim 42 , wherein the matrix is repeatedly expanded and additional oligonucleotides are hybridized to the chromosome and attached to the matrix. 
     
     
         58 . The method of  claim 42 , wherein each of the plurality of oligonucleotides is directly attached to the matrix. 
     
     
         59 . The method of  claim 42 , wherein each of the plurality of oligonucleotides is indirectly attached to the matrix. 
     
     
         60 . The method of  claim 42 , wherein each of the plurality of oligonucleotides comprises a second non-complementary region, the method comprising amplifying and/or sequencing at least a portion of the second non-complementary region. 
     
     
         61 . The method of  claim 60 , wherein amplification or sequencing comprises the use of a nucleotide comprising a detectable label.