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 . A random array of concatemers, wherein said random array comprises:
 (a) a solid substrate comprising a surface, wherein said surface comprises a plurality of discrete spaced apart regions defined on said surface, wherein said discrete spaced apart regions are separated by inter-regional areas and, wherein each of said discrete regions has an area;   (b) a population of concatemers randomly disposed on said plurality of discrete regions such that at least a majority of said plurality of discrete regions comprises a single concatemer attached thereto and said inter-regional areas are substantially free of concatemers, wherein said concatemers are generated from a circular template and comprise a plurality of monomeric units, and wherein each monomeric unit comprises:
 (i) a first target sequence of a target polynucleotide, and 
 (ii) an adaptor, 
   and wherein said concatemers have a diameter that is approximately equal to that of said discrete spaced apart regions,   and wherein said population of concatemers comprises a plurality of different target polynucleotides.   
     
     
         2 . The random array of  claim 1 , wherein said target polynucleotide is genomic DNA. 
     
     
         3 . The random array of  claim 2 , wherein said genomic DNA is human genomic DNA. 
     
     
         4 . The random array of  claim 1 , wherein said concatemers are non-covalently immobilized to said discrete regions. 
     
     
         5 . The random array of  claim 1 , wherein said substrate comprises silica. 
     
     
         6 . The random array of  claim 1 , wherein said discrete regions are arranged on said surface in a pattern. 
     
     
         7 . The random array of  claim 1 , wherein said circular template comprises said adaptor and said first target sequence. 
     
     
         8 . The random array of  claim 1 , wherein said discrete regions have a nearest neighbor distance of 200 nm or greater. 
     
     
         9 . The random array of  claim 1 , wherein said discrete regions comprise an amino group. 
     
     
         10 . The random array of  claim 1 , wherein said area of said discrete spaced apart regions is less than 1 μm 2 . 
     
     
         11 . The random array of  claim 1 , wherein said area of said discrete spaced apart regions is in the range of from 0.04 μm 2  to 1 μm 2 . 
     
     
         12 . The random array of  claim 1 , wherein a majority of said concatemers have a size in the range of 50-250 kb. 
     
     
         13 . The random array of  claim 1 , wherein said discrete regions are prepared by photolithography. 
     
     
         14 . A random array of concatemers, wherein said random array comprises:
 (a) a solid substrate comprising a surface, wherein said surface comprises a plurality of discrete spaced apart regions defined on said surface, wherein said discrete spaced apart regions are separated by inter-regional areas, wherein each of said discrete regions has an area;   (b) a population of concatemers randomly disposed on said plurality of discrete regions such that at least a majority of said plurality of discrete regions comprises a single concatemer attached thereto, wherein said concatemers are generated from a circular template, wherein said inter-regional areas are substantially free of concatemers, and wherein said concatemers comprise a plurality of monomeric units and each monomeric unit comprises:
 (i) a first target sequence of a target polynucleotide, and 
 (ii) an adaptor, 
   wherein each of said discrete regions comprise a number of reactive functionalities, and   wherein said number is such that a single concatemer occupies substantially all of said number of reactive functionalities, and   wherein said population of concatemers comprises a plurality of different target polynucleotides.   
     
     
         15 . A random array of concatemers, wherein said random array comprises:
 (a) a solid substrate comprising a surface, wherein said surface comprises a plurality of discrete spaced apart regions defined on said surface, wherein said discrete spaced apart regions are separated by inter-regional areas, wherein each of said discrete regions has an area;   (b) a population of concatemers randomly disposed on said plurality of discrete regions, wherein each of said discrete regions has an area corresponding in size to that of said concatemers such that at least a majority of said plurality of discrete regions comprises a single concatemer attached thereto, wherein said inter-regional areas are substantially free of concatemers, and wherein said concatemers comprise a plurality of monomeric units and each monomeric unit comprises:
 (i) a first target sequence of a target polynucleotide, and 
 (ii) an adaptor, 
   wherein said population of concatemers comprises a plurality of different target polynucleotides.