Single Molecule Arrays for Genetic and Chemical Analysis

Abstract

Random arrays of single molecules are provided for carrying out large scale analyses, particularly of biomolecules, such as genomic DNA, cDNAs, proteins, and the like. In one aspect, arrays of the invention comprise concatemers of DNA fragments that are randomly disposed on a regular array of discrete spaced apart regions, such that substantially all such regions contain no more than a single concatemer. Preferably, such regions have areas substantially less than 1 μm 2 and have nearest neighbor distances that permit optical resolution of on the order of 10 9 single molecules per cm 2 . Many analytical chemistries can be applied to random arrays of the invention, including sequencing by hybridization chemistries, sequencing by synthesis chemistries, SNP detection chemistries, and the like, to greatly expand the scale and potential applications of such techniques.

1 . An array of single molecules comprising:
 a support having a planar surface having a regular array of discrete spaced apart regions, wherein each discrete spaced apart region has an area of less than 1 μm 2  and contains reactive functionalities attached thereto; and
 a plurality of single molecules attached to the surface, wherein each single molecule comprises
 (i) a macromolecular structure and 
 (ii) at least one analyte having an attachment moiety, 
 
 such that each macromolecular structure comprises a unique functionality and a plurality of attachment functionalities that are capable of forming linkages with the reactive functionalities of the discrete spaced apart regions, 
 and such that the analyte is attached to the macromolecular structure by a linkage between the unique functionality and the attachment moiety of the analyte; 
 wherein each of said discrete spaced apart regions is surrounded by an inter-regional space that is substantially free of said macromolecular structures; and 
 wherein the plurality of single molecules are randomly disposed on the discrete spaced apart regions such that at least a majority of the discrete spaced apart regions contain only one single molecule comprising a macromolecular structure and at least one analyte. 
   
     
     
         2 . The array of  claim 1  wherein said macromolecular structure is a branched or linear polymer. 
     
     
         3 . The array of  claim 2  wherein said discrete spaced apart regions are wells in said support, the wells each having an opening with an area equal to or less than that of said discrete spaced apart regions. 
     
     
         4 . The array of  claim 2  wherein said discrete spaced apart regions each have a nearest neighbor distance in the range of from 0.1 to 20 μm and wherein said macromolecular structures are branched or linear polynucleotides. 
     
     
         5 . The array of  claim 4  wherein said nearest neighbor distance is in the range of from 0.3 to 3 μm. 
     
     
         6 . The array of  claim 5  wherein substantially every said discrete spaced apart region has a single molecule attached. 
     
     
         7 . The array of  claim 5  wherein said macromolecular structures are linear polynucleotides. 
     
     
         8 . The array of  claim 5  wherein said discrete spaced apart regions are disposed on said planar surface in a rectilinear pattern wherein each discrete spaced apart region has an area enclosed by a circle having a diameter of from 0.1 to 10 μm. 
     
     
         9 . The array of  claim 5  wherein said analyte is a polynucleotide or a protein. 
     
     
         10 . The array of  claim 5  wherein said unique functionality is a first oligonucleotide and wherein said attachment moiety is a second oligonucleotide having a sequence complementary to the first oligonucleotide. 
     
     
         11 . The array of  claim 10  wherein said analyte is a polynucleotide. 
     
     
         12 . A kit for constructing a single molecule array, the kit comprising:
 a support having a surface having reactive functionalities; and   a plurality of macromolecular structures each having a unique functionality and multiple complementary functionalities, the macromolecular structures being capable of being attached randomly on the surface wherein the attachment is formed by one or more linkages formed by reaction of one or more reactive functionalities with one or more complementary functionalities; and wherein the unique functionality is capable of selectively reacting with a functionality on an analyte molecule to form the single molecule array.   
     
     
         13 . The kit of  claim 12  wherein said surface is a planar surface having an array of discrete spaced apart regions containing said reactive functionalities and wherein each discrete spaced apart region has an area less than 1 μm 2 . 
     
     
         14 . The kit of  claim 13  wherein said discrete spaced apart regions form a regular array with a nearest neighbor distance in the range of from 0.1 to 20 μm. 
     
     
         15 . The array of  claim 1  wherein the macromolecular structure provides multivalent attachment of the single molecule to the surface via attachment functionalities that form covalent linkages with the reactive functionalities. 
     
     
         16 . The array of  claim 1  wherein the macromolecular structure provides multivalent attachment of the single molecule to the surface via attachment functionalities that form noncovalent bonds with the reactive functionalities. 
     
     
         17 . The array of  claim 1  wherein the discrete spaced apart regions are approximately circular or square in shape so that their sizes can be indicated by a single linear dimension, and the size of such regions are in the range of from 125 nm to 250 nm, or in the range of from 200 nm to 500 nm. 
     
     
         18 . The array of  claim 1  wherein the regular array comprises a rectilinear pattern. 
     
     
         19 . A method of making an array of  claim 1  comprising
 (i) attaching a plurality of branched macromolecular structures to discrete spaced apart regions on a support, said support having a planar surface having a regular array of discrete spaced apart regions, wherein each discrete spaced apart region has an area of less than 1 μm 2 , where the attaching comprises a linkage between reactive functionalities of the discrete spaced apart regions and attachment functionalities of the branched macromolecular structures and 
 (ii) attaching single analyte molecules to individual branched macromolecular structures via a linkage between an attachment moiety of the analyte and a unique functionality of the branched macromolecular structure. 
 
     
     
         20 . The method of  claim 19  wherein the branched macromolecular structures are non-covalently attached to discrete spaced apart regions on a support and the single analyte molecules are attached to individual branched macromolecular structures. 
     
     
         21 . The method of  claim 19  wherein step (i) is carried out before step (ii). 
     
     
         22 . The method of  claim 19  wherein step (ii) is carried out before step (i).