Single molecule arrays for genetic and chemical analysis
First Claim
1. A method of identifying a sequence of a target polynucleotide, said method comprising:
- a) providing a substrate comprising a plurality of discrete regions, wherein a plurality of concatemers are randomly disposed on said plurality of said discrete regions, wherein at least a majority of said discrete regions comprises a single concatemer, wherein at least a majority of said concatemers at said plurality of discrete regions are optically resolvable, andwherein each concatemer comprises a plurality of monomeric units and each monomeric unit comprises;
i) a first target sequence of said target polynucleotide;
ii) an adaptor comprising a Type IIs endonuclease restriction site, wherein said first target sequence is adjacent to said adaptor;
b) applying a first set of probes to said substrate such that one or more probes from said first set hybridizes to said adaptor;
c) applying a second set of probes to said substrate such that one or more probes from said second set hybridizes to said first target sequence;
d) ligating probes from said first set and probes from said second set that are hybridized to adjacent sequences of said monomeric unit to form a ligated complex;
e) detecting said ligated complex thereby identifying a nucleotide of said target polynucleotide;
f) stripping off said ligated complex;
g) repeating steps (b) through (f) in order to determine said sequence of said target polynucleotide.
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Accused Products
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 μm2 and have nearest neighbor distances that permit optical resolution of on the order of 109 single molecules per cm2. 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.
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Citations
20 Claims
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1. A method of identifying a sequence of a target polynucleotide, said method comprising:
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a) providing a substrate comprising a plurality of discrete regions, wherein a plurality of concatemers are randomly disposed on said plurality of said discrete regions, wherein at least a majority of said discrete regions comprises a single concatemer, wherein at least a majority of said concatemers at said plurality of discrete regions are optically resolvable, and wherein each concatemer comprises a plurality of monomeric units and each monomeric unit comprises; i) a first target sequence of said target polynucleotide; ii) an adaptor comprising a Type IIs endonuclease restriction site, wherein said first target sequence is adjacent to said adaptor; b) applying a first set of probes to said substrate such that one or more probes from said first set hybridizes to said adaptor; c) applying a second set of probes to said substrate such that one or more probes from said second set hybridizes to said first target sequence; d) ligating probes from said first set and probes from said second set that are hybridized to adjacent sequences of said monomeric unit to form a ligated complex; e) detecting said ligated complex thereby identifying a nucleotide of said target polynucleotide; f) stripping off said ligated complex; g) repeating steps (b) through (f) in order to determine said sequence of said target polynucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A method of identifying a sequence of a target polynucleotide, said method comprising:
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a) providing a substrate comprising a plurality of discrete regions, wherein a plurality of concatemers are randomly disposed on said plurality of discrete regions, wherein at least a majority of said discrete regions comprises a single concatemer disposed thereon, wherein at least a majority of said concatemers at said plurality of discrete regions are optically resolvable, and wherein each concatemer comprises a plurality of monomeric units and each monomeric unit comprises; i) a first target sequence of said target polynucleotide; ii) an adaptor, wherein said first target sequence is adjacent to said adaptor; b) applying a first set of probes to said substrate such that one or more probes from said first set hybridizes to said adaptor; c) applying a second set of probes to said substrate such that one or more probes from said second set hybridizes to said first target sequence; d) ligating probes from said first set and probes from said second set that are hybridized to adjacent sequences of said monomeric unit to form a ligated complex; e) detecting said ligated complex; thereby identifying said sequence of said target polynucleotide. - View Dependent Claims (12, 13, 14, 15, 16, 17)
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18. A method of identifying a sequence of a target polynucleotide, said method comprising:
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a) providing a substrate comprising a plurality of discrete regions, wherein a plurality of concatemers are randomly disposed on said plurality of said discrete regions, wherein a at least majority of said discrete regions comprises a single concatemer, wherein said substrate further comprises inter-regional areas between said discrete regions, wherein said concatemers do not bind to said inter-regional areas, wherein at least a majority of said concatemers at said plurality of discrete regions are optically resolvable, and wherein each concatemer comprises a plurality of monomeric units and each monomeric unit comprises; (i) a first target sequence of said target polynucleotide; (ii) an adaptor, wherein said first target sequence is adjacent to said adaptor; b) applying a first set of probes to said substrate such that one or more probes from said first set hybridizes to said adaptor; c) applying a second set of probes to said substrate such that one or more probes from said second set hybridizes to said first target sequence; d) ligating probes from said first set and probes from said second set that are hybridized to adjacent sequences of said monomeric unit to form a ligated complex; e) detecting said ligated complex, thereby identifying a nucleotide of said target polynucleotide; f) stripping off said ligated complex; g) repeating steps (b) through (f) in order to determine said sequence of said target polynucleotide. - View Dependent Claims (19, 20)
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Specification