Dna array

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.

1 . An array of polymer molecules comprising: a support having a surface; and a plurality of polymer molecules attached to the surface, wherein each polymer molecule has a random coil state and comprises a branched or linear structure of multiple copies of one or more linear polymeric units, such that the polymer molecule is attached to the surface within a region substantially equivalent to a projection of the random coil on the surface and randomly disposed at a density such that at least thirty percent of the polymer molecules are separately detectable. 
     
     
         2 . The array of polymer molecules of  claim 1  wherein said one or more linear polymeric units are each single stranded polynucleotides and wherein said surface has reactive functionalities or capture oligonucleotides attached thereto and wherein said polymer molecules are each attached to said surface by one or more linkages formed by one or more reactive functionalities reacting with complementary functionalities of said polymers molecules or by one or more complexes formed between the capture oligonucleotides and complementary sequences of the polymer molecules. 
     
     
         3 . The array of  claim 2  wherein said surface is a planar surface having an array of discrete spaced apart regions, wherein each discrete spaced apart region has a size substantially equivalent to said projection of said random coil of said polymer molecule and contains said reactive functionalities or said capture oligonucleotides attached thereto. 
     
     
         4 . The array of  claim 3  wherein each of said discrete spaced apart regions has an area of less than 1 0.1 to 20 μm 2 . 
     
     
         5 . The array of  claim 4  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 and wherein a majority of said discrete spaced apart regions contain no more than one said polymer molecules. 
     
     
         6 . The array of  claim 5  wherein said polymer molecules are randomly distributed on said discrete spaced apart regions and wherein said nearest neighbor distance is in the range of from 0.3 to 3 μm. 
     
     
         7 . The array of  claim 6  wherein each of said polymer molecules is a polynucleotide molecule comprising a concatemer of multiple copies of a target sequence and an adaptor oligonucleotide. 
     
     
         8 . The array of  claim 4  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. 
     
     
         9 . An array of polynucleotide molecules comprising: a support having a surface; and a plurality of polynucleotide molecules attached to the surface, wherein each polynucleotide molecule has a random coil state and comprises a concatemer of multiple copies of a target sequence such that the polynucleotide molecule is attached to the surface within a region substantially equivalent to a projection of the random coil on the surface and randomly disposed at a density such that at least thirty percent of the polynucleotide molecules have a nearest neighbor distance of at least fifty nm. 
     
     
         10 . The array of  claim 9  wherein said surface has reactive functionalities attached thereto and wherein said polynucleotide molecules are each attached to said surface by one or more linkages formed by one or more reactive functionalities reacting with complementary functionalities of said polynucleotide molecules. 
     
     
         11 . The array of  claim 10  wherein said surface is a planar surface having an array of discrete spaced apart regions, wherein each discrete spaced apart region has a size substantially equivalent to said projection of said random coil of said polynucleotide molecule and contains said reactive functionalities attached thereto and wherein such regions have at most one of said polynucleotides attached. 
     
     
         12 . The array of  claim 11  wherein said reactive functionalities are hydrophobic functionalities. 
     
     
         13 . The array of  claim 11  wherein said discrete spaced apart region has an area of less than 1 μm 2 . 
     
     
         14 . The array of  claim 13  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. 
     
     
         15 . The array of  claim 13  wherein said polynucleotides are randomly distributed on said discrete spaced apart regions and wherein said nearest neighbor distance is in the range of from 0.3 to 3 μm. 
     
     
         16 . The array of  claim 15  wherein substantially every said discrete spaced apart region has a polynucleotide attached. 
     
     
         17 . The array of  claim 15  wherein said concatemer comprises alternating copies of said target sequence and said adaptor oligonucleotide. 
     
     
         18 . The array of  claim 17  wherein each of said concatemers contains at least 10 copies of its respective target sequence. 
     
     
         19 . The array of  claim 17  wherein said target sequences each have a length in the range of from 50 to 500 nucleotides and wherein said adaptor oligonucleotide has a length in the range of from 6 to 60 nucleotides. 
     
     
         20 - 69 . (canceled) 
     
     
         70 . A method of determining a nucleotide sequence of a target polynucleotide, the method comprising the steps of: (a) generating a plurality of target concatemers from the target polynucleotide, each target concatemer comprising multiple copies of a fragment of the target polynucleotide and the plurality of target concatemers including a number of fragments that substantially covers the target polynucleotide; (b) forming a random array of target concatemers fixed to a surface at a density such that at least a majority of the target concatemers are optically resolvable; (c) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (d) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (e) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites; (f) identifying the sequences of the ligated first and second probes; and (g) repeating steps (c) through (f) until the sequence of the target polynucleotide can be determined from the identities of the sequences of the ligated probes. 
     
     
         71 - 73 . (canceled)