Novel oligonucleotide arrays and their use for sorting, isolating, sequencing, and manipulating nucleic acids
0 Assignments
0 Petitions
Accused Products
Abstract
A method of sorting mixtures of nucleic acid strands comprising hybridizing the strands to an array of immobilized oligonucleotides, each of which includes a constant segment adjacent to a variable segment. The constant segment of the immobilized oligonucleotides can be made complementary to the ends of strands obtained by digesting a double-stranded nucleic acid with a restriction enzyme and restoring the restriction sites, thereby permitting the sorting of strands according to their variable sequences adjacent to their constant terminal restored restriction sites.
81 Citations
189 Claims
-
1-159. -159. (canceled)
-
160. A method of analyzing a nucleic acid, comprising:
-
amplifying a mixture of nucleic acids comprising a group of genome fragments by a method comprising; (i) cleaving a genomic DNA sample with a restriction enzyme, thereby providing restriction fragments, wherein a plurality of the fragments comprise two complementary individual strands; (ii) ligating adaptor nucleic acids to the restriction fragments, wherein said adaptor nucleic acids comprise a first universal priming region, thereby providing adaptor-ligated fragments; (iii) hybridizing the adaptor-ligated fragments to immobilized oligonucleotides wherein said immobilized oligonucleotides are attached to a solid support and comprise a region that is complementary to said first universal priming region and a 3′
end available for extension, wherein individual strands hybridize to immobilized oligonucleotides to form hybridized target strands;(iv) extending the immobilized oligonucleotides using the hybridized target strands as template, thereby providing extended immobilized oligonucleotides comprising a complimentary copy of the hybridized target strands; and (v) amplifying the extended immobilized oligonucleotides to obtain multiple complementary copies of said hybridized target strands, wherein said copies are attached to said solid support, thereby providing an amplified nucleic acid mixture of genome fragments; and analyzing at least one nucleic acid of interest in said amplified mixture. - View Dependent Claims (161)
-
-
162. A method of analyzing at least one nucleic acid, comprising:
-
obtaining a plurality of amplified genomic fragments separated into discreet features of an array by a method comprising; (a) fragmenting a genomic DNA sample comprising at least one nucleic acid, thereby providing fragments; (b) ligating an adaptor to the fragments to generate adaptor-ligated fragments, wherein said adaptor comprises a universal priming sequence; (c) providing an oligonucleotide array comprising oligonucleotides that are complementary to the universal priming sequence in the adaptor, wherein said oligonucleotides are attached to a solid support; (d) hybridizing the adaptor-ligated fragments to the oligonucleotides on the solid support so that adaptor-ligated fragments of different sequence are hybridized at different discreet locations of the solid support; (e) amplifying the adaptor-ligated fragments by extending the oligonucleotides using a DNA polymerase to obtain immobilized extended polynucleotides of different sequences and amplifying the immobilized extended polynucleotides, thereby providing an array of amplified genomic fragments of different sequences present in different discreet locations of an array; and (f) analyzing at least one of the amplified genomic fragments. - View Dependent Claims (163, 164, 165, 166, 167, 168, 169, 170)
-
-
171. A method for analyzing a plurality of different nucleic acid sequences in a complex nucleic acid sequence comprising:
-
(a) fragmenting said complex nucleic acid sample to obtain a plurality of different sequence nucleic acid fragments, said fragments comprising complimentary fragment strands; (b) attaching a first adaptor sequence to the 5′
ends of the fragment strands and a second adaptor sequence to the 3′
ends of the fragment strands, to obtain a plurality of different sequence, adaptor-modified fragments having a first universal priming sequence at the 5′
ends of the strands and a second universal priming sequence at the 3′
end of the strands;(c) hybridizing the adaptor-modified fragments to an array of oligonucleotides attached to a solid support wherein said oligonucleotides are attached to the solid support at the 5′
end and have a free 3′
end, and wherein said oligonucleotides comprise a sequence that is complementary to the second universal priming sequence;(d) extending the oligonucleotides with a polymerase using the adaptor-modified fragments as template to obtain extended oligonucleotides that comprise at their 3′
ends the complement of the first universal priming sequence;(e) amplifying the extended oligonucleotides to obtain a plurality of copies of each different nucleic acid sequence by hybridizing a primer to the extended oligonucleotides, wherein said primer comprises said first universal priming sequence and extending said primer to obtain a copy of said extended oligonucleotides and amplifying the copy of the extended oligonucleotides to obtain a plurality of copies of each different nucleic acid sequence; and (f) analyzing the plurality of different nucleic acid sequences. - View Dependent Claims (172, 173, 174, 175)
-
-
176. A method for generating an array of immobilized copies of a plurality of nucleic acids for analysis, comprising:
-
(a) obtaining a sample comprising said plurality of nucleic acids; (b) fragmenting the sample to obtain target fragments comprising said plurality of nucleic acids, wherein said target fragments each comprise a first fragment strand and a second fragment strand; (c) adding a first constant sequence to the 3′
termini of the first fragment strands and a second constant sequence to the 5′
termini of the first fragment strands wherein said first constant sequence comprises a first priming sequence and said second constant sequence comprises a second priming sequence;(d) hybridizing the fragment strands from (c) to a solid support comprising immobilized oligonucleotides complementary to the first priming sequence and having free 3′
ends, thereby forming complexes between the fragment strands and the immobilized oligonucleotides; and(e) extending the immobilized oligonucleotides using the hybridized fragment strands as template to obtain immobilized complementary copies of the hybridized fragment strands. - View Dependent Claims (177, 178, 179, 180, 181, 182, 183, 184, 185, 186)
-
-
187. A method for analyzing target nucleic acids in a nucleic acid sample comprising:
-
fragmenting the nucleic acid sample to obtain nucleic acid fragments comprising target nucleic acids, wherein said fragments comprise a first strand and a second strand; adding a first universal priming sequence to the 3′
end of the first strand of a plurality of the fragments to obtain first strands having said first universal priming region at the 3′
ends;hybridizing said strands having said universal priming region at the 3′
ends to a plurality of support bound oligonucleotides to obtain hybridized strands, wherein said support bound oligonucleotides have a free 3′
termini and comprise a constant region that is complementary to said universal priming region;extending the support bound oligonucleotides from the free 3′
termini using the hybridized strands as template, in a reaction comprising DNA polymerase, to obtain extended oligonucleotides comprising a complimentary copy of a hybridized strand;amplifying said complimentary copy of the hybridized strand by PCR to obtain a plurality of complementary copies of the hybridized strand; and analyzing the plurality of complementary copies of the hybridized strand to determine the sequence of target nucleic acids. - View Dependent Claims (188, 189)
-
Specification