Nucleic acid arrays and methods of synthesis
First Claim
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1. A method of generating an array, comprising:
- (a) providing;
i) a solid support comprising a plurality of positions for oligonucleotides, said positions defined by x, and y coordinates;
ii) a plurality of identical oligonucleotides, each oligonucleotide comprising a sequence; and
iii) a plurality of unique circular DNA templates, each circular DNA template comprising a sequence of interest and a region complementary to at least a portion of said sequence of said oligonucleotides, said sequence of interest being different for each circular template;
(b) immobilizing one oligonucleotide from said plurality of identical oligonucleotides in each of said positions on said solid support to create an ordered array comprising a plurality of identical immobilized oligonucleotides;
(c) adding to each immobilized oligonucleotide of said ordered array a circular DNA template from said plurality of said unique circular DNA templates under conditions such that said immobilized oligonucleotide hybridizes to said circular DNA template to create a plurality of primed circular templates, each primed circular template comprising a different sequence of interest; and
(d) extending each of said primed circular templates along a z coordinate to create an extended immobilized oligonucleotide comprising at least two copies of said sequence of interest, thereby generating an ordered redundant array, wherein said ordered redundant array refers to said array having at least two copies of said sequence of interest along the z coordinate.
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Abstract
The present invention generally relates to high density nucleic acid arrays and methods of synthesizing nucleic acid sequences on a solid surface. Specifically, the present invention contemplates the use of stabilized nucleic acid primer sequences immobilized on solid surfaces, and circular nucleic acid sequence templates combined with the use of isothermal rolling circle amplification to thereby increase nucleic acid sequence concentrations in a sample or on an array of nucleic acid sequences.
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Citations
22 Claims
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1. A method of generating an array, comprising:
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(a) providing;
i) a solid support comprising a plurality of positions for oligonucleotides, said positions defined by x, and y coordinates;
ii) a plurality of identical oligonucleotides, each oligonucleotide comprising a sequence; and
iii) a plurality of unique circular DNA templates, each circular DNA template comprising a sequence of interest and a region complementary to at least a portion of said sequence of said oligonucleotides, said sequence of interest being different for each circular template;
(b) immobilizing one oligonucleotide from said plurality of identical oligonucleotides in each of said positions on said solid support to create an ordered array comprising a plurality of identical immobilized oligonucleotides;
(c) adding to each immobilized oligonucleotide of said ordered array a circular DNA template from said plurality of said unique circular DNA templates under conditions such that said immobilized oligonucleotide hybridizes to said circular DNA template to create a plurality of primed circular templates, each primed circular template comprising a different sequence of interest; and
(d) extending each of said primed circular templates along a z coordinate to create an extended immobilized oligonucleotide comprising at least two copies of said sequence of interest, thereby generating an ordered redundant array, wherein said ordered redundant array refers to said array having at least two copies of said sequence of interest along the z coordinate. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
(a) providing i) an ordered redundant array of immobilized oligonucleotides produced according to the method of claim 1, wherein said array comprises a plurality of identical immobilized oligonucleotides, wherein each immobilized oligonucleotide has a circular DNA containing a sequence of interest that has been extended along the z coordinate to have at least two copies of said sequence of interest; and
ii) a plurality of fragments of a target nucleic acid; and
(b) bringing said fragments of said target nucleic acid into contact with said array under conditions such that at least one of said fragments hybridizes to one of said extended immobilized oligonucleotides on said array.
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12. A method of generating an array capable of hybridizing to fragments of a target nucleic acid, comprising:
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a) providing;
i) a solid support comprising positions for oligonucleotides, said positions defined by x and y coordinates;
ii) a plurality of oligonucleotides, each oligonucleotide comprising a sequence complementary to a different portion of the sequence of said target nucleic acid; and
iii) a plurality of corresponding circular DNA templates, each circular DNA template comprising a different portion of the sequence of said target;
b) immobilizing each of said oligonucleotides in one of said positions on said solid support to create an ordered array comprising a plurality of immobilized oligonucleotides;
c) adding to each immobilized oligonucleotide of said ordered array along a z coordinate a corresponding circular DNA template under conditions such that said immobilized oligonucleotide hybridizes to said corresponding circular DNA template to create a plurality of primed circular templates; and
d) extending said primed circular templates to create an ordered redundant array of extended immobilized oligonucleotides, each extended immobilized oligonucleotide comprising at least two copies of said portion of said sequence of said target nucleic acid, wherein said ordered redundant array refers to said array having at least two copies along the z coordinate of said portion of the sequence of interest contained in said primed circular template. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
a) providing i) an array capable of hybridizing to fragments of a target nucleic acid produced according to the method of claim 12; and
ii) a plurality of fragments of a target nucleic acid; and
b) bringing said fragments of said target nucleic acid into contact with said array under conditions such that at least one of said fragments hybridizes to one of said extended immobilized oligonucleotides on said array.
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Specification