Replica amplification of nucleic acid arrays
First Claim
1. A method of producing a plurality of a nucleic acid array, comprising, in order, the steps of,a) providing a first nucleic acid array comprising a pool of nucleic acid molecules immobilized to a support with the nucleic acid molecules being randomly patterned, and amplifying in situ the nucleic acid molecules, b) transferring at least a subset of nucleic acid molecules produced by said amplifying is to a second support, and c) immobilizing said subset to said second support to form a second nucleic acid array, wherein the nucleic acid molecules of said second array occupy positions that correspond to those of said nucleic acid molecules from which they were amplified on said first array to produce said plurality.
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Abstract
A method of producing a plurality of a nucleic acid array, comprising, in order, the steps of amplifying in situ nucleic acid molecules of a first randomly-patterned, immobilized nucleic acid array comprising a heterogeneous pool of nucleic acid molecules affixed to a support, transferring at least a subset of the nucleic acid molecules produced by such amplifying to a second support, and affixing the subset so transferred to the second support to form a second randomly-patterned, immobilized nucleic acid array, wherein the nucleic acid molecules of the second array occupy positions that correspond to those of the nucleic acid molecules from which they were amplified on the first array, so that the first array serves as a template to produce a plurality, is disclosed.
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Citations
39 Claims
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1. A method of producing a plurality of a nucleic acid array, comprising, in order, the steps of,
a) providing a first nucleic acid array comprising a pool of nucleic acid molecules immobilized to a support with the nucleic acid molecules being randomly patterned, and amplifying in situ the nucleic acid molecules, b) transferring at least a subset of nucleic acid molecules produced by said amplifying is to a second support, and c) immobilizing said subset to said second support to form a second nucleic acid array, wherein the nucleic acid molecules of said second array occupy positions that correspond to those of said nucleic acid molecules from which they were amplified on said first array to produce said plurality.
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21. A plurality of a nucleic acid array, wherein said plurality comprises
a) a first template randomly-patterned, covalently immobilized nucleic acid array comprising a pool of nucleic acid molecules randomly immobilized on a support, and b) a second randomly-patterned, covalently immobilized nucleic acid array, wherein the nucleic acid molecules of said second array are nucleic acid amplification products of said pool and wherein said nucleic acid molecules of said second array occupy positions on said second array that correspond to those of said nucleic acid molecules from which they were amplified on said first array.
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22. A method for determining the sequential order of genetic elements of a chromosome, comprising providing an immobilized nucleic acid array, comprising the steps of
a) providing an immobilized chromosome, b) amplifying the nucleic acid sequences of said chromosome, c) contacting said amplified sequences with a support, such that a subset of nucleic acid molecules produced by said amplifying are retained by said support, d) immobilizing said nucleic acid molecules so retained to said support to form a first immobilized nucleic acid array, wherein the positions of the members of said array correspond to the positions of the DNA sequences from which they were amplified on said chromosome, and e) determining the order of genetic elements on said chromosome, wherein said ordering comprises identifying the features of said array and wherein the position of a first feature relative that of a second feature on said array corresponds to the position of a first genetic element relative to that of a second genetic element on said chromosome.
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25. A method for localizing RNA molecules within a cell or a tissue section comprising providing an immobilized nucleic acid array, comprising the steps of
a) providing an immobilized cell or a tissue section, b) reverse transcribing RNA molecules of said cell or said tissue section to produce an array of features comprising reverse transcripts, c) contacting said array with a support, such that at least a subset of said reverse transcripts are retained by said support, and d) immobilizing said reverse transcripts to said support to form an immobilized nucleic acid array, and e) localizing said RNA molecules, comprising identifying said features of said array, wherein the positions of said features on said array correspond to the positions of said RNA molecules in said cell or said tissue section.
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30. A method of obtaining a plurality of immobilized nucleic acid arrays, wherein the arrays of the plurality are derived from different nucleic acid pools, comprising the steps of
a) providing a first immobilized nucleic array acid comprising a first pool of nucleic acid molecules that have linked to both ends oligonucleotide sequences each comprising a restriction enzyme(s) recognition site, such that cleavage of the nucleic acid molecules of said pool with said enzyme(s) results in the release of pairs of oligonucleotide primers that comprise sequences unique to either end of each member of said pool, b) amplifying by PCR the nucleic acid molecules of said array, c) contacting said first immobilized nucleic acid array with a support such that at least a subset of nucleic acid molecules produced by said amplifying are transferred to said support, d) immobilizing said nucleic acid molecules to said support to form a replica of said first immobilized nucleic acid array, wherein the positions of said nucleic acid molecules on said replica correspond to the positions of said nucleic acid molecules of said first array from which they were amplified, e) cleaving said nucleic acid molecules of said replica with said restriction enzyme(s), thereby forming an array of immobilized oligonucleotide primers that comprise sequences unique to either end of each feature of said first nucleic acid array, f) washing from said oligonucleotide primers the nucleic acid fragments released from them by said cleaving, g) contacting said primers with a second pool of nucleic acid molecules under conditions that permit hybridization of the nucleic acid molecules that are complementary, such that hybridization occurs between said oligonucleotide primers and the nucleic acid molecules of said second nucleic acid pool, h) amplifying the nucleic acid molecules of said second pool so hybridized to said primers, wherein said immobilized oligonucleotide primers to which they are hybridized serve to prime said amplifying, thereby forming an immobilized array of nucleic acid molecules of said second pool.
Specification