Self-assembled single molecule arrays and uses thereof
First Claim
1. A method of determining sequence information for a target polynucleotide, comprising:
- (a) providing a plurality of concatemers, each concatemer having been obtained by;
(i) forming a first circular DNA comprising a fragment of said target polynucleotide and a first adaptor, wherein the first adaptor comprises a binding site for a restriction enzyme that cleaves DNA at a cleavage site separated from said binding site by at least six nucleotides;
(ii) forming a linearized DNA by a process that comprises cleaving the first circular DNA at a site that is internal to the target polynucleotide using a restriction endonuclease that binds to said binding site, whereby the linearized DNA comprises the first adaptor flanked on each side by a portion of said fragment;
(iii) forming a second circular DNA comprising said linearized DNA and a second adaptor;
(iv) amplifying the second circular DNA to form said concatemer;
(b) forming an array of said concatemers bound to a surface; and
(c) determining target sequences both upstream and downstream from the second adaptor by a process that comprises hybridizing oligonucleotides to the second adaptor in a plurality of the concatemers in the array.
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Abstract
The present invention provides methods of making and using self-assembled arrays of single polynucleotide molecules for carrying out a variety of large-scale genetic measurements, such as gene expression analysis, gene copy number assessment, and the like. Random arrays used in the invention are “self-assembled” in the sense that they are formed by deposition of polynucleotide molecules onto a surface where they become fixed at random locations. The polynucleotide molecules fixed on the surface are then identified by direct sequence determination of component nucleic acids, such as incorporated probe sequences, or by other decoding schemes. Such identification converts a random array of determinable polynucleotides, and their respective probes into an addressable array of probe sequences.
149 Citations
15 Claims
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1. A method of determining sequence information for a target polynucleotide, comprising:
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(a) providing a plurality of concatemers, each concatemer having been obtained by; (i) forming a first circular DNA comprising a fragment of said target polynucleotide and a first adaptor, wherein the first adaptor comprises a binding site for a restriction enzyme that cleaves DNA at a cleavage site separated from said binding site by at least six nucleotides; (ii) forming a linearized DNA by a process that comprises cleaving the first circular DNA at a site that is internal to the target polynucleotide using a restriction endonuclease that binds to said binding site, whereby the linearized DNA comprises the first adaptor flanked on each side by a portion of said fragment; (iii) forming a second circular DNA comprising said linearized DNA and a second adaptor; (iv) amplifying the second circular DNA to form said concatemer; (b) forming an array of said concatemers bound to a surface; and (c) determining target sequences both upstream and downstream from the second adaptor by a process that comprises hybridizing oligonucleotides to the second adaptor in a plurality of the concatemers in the array. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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Specification