Novel oligonucleotide arrays and their use for sorting, isolating, sequencing, and manipulating nucleic acids

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.

1 - 159 . (canceled) 
     
     
         160 . A method of analyzing a nucleic acid, comprising:
 providing at least one oligonucleotide which is complementary to a target sequence of interest in a genomic DNA sample;   amplifying a mixture of nucleic acids comprising a group of genome fragments by a method comprising:   cleaving a genomic DNA sample with a restriction enzyme, thereby providing restriction fragments;   ligating adaptor nucleic acids to the restriction fragments, thereby providing adaptor-ligated fragments;   hybridizing the adaptor-ligated fragments to immobilized oligonucleotides that are complementary to the adaptor nucleic acids wherein the immobilized oligonucleotides are attached to a solid support, and extending the hybridized immobilized oligonucleotides using the adaptor-ligated fragments as template, thereby providing extended immobilized oligonucleotides; and   amplifying the extended immobilized oligonucleotides, thereby providing an amplified nucleic acid mixture comprising genome fragments; and   hybridizing the at least one oligonucleotide to the amplified nucleic acid mixture, thereby analyzing at least one nucleic acid of interest in the amplified mixture.   
     
     
         161 . The method of  claim 160 , wherein the oligonucleotide is a member of an array of oligonucleotides, which array comprises additional oligonucleotides which hybridize to different target sequences of interest. 
     
     
         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 the oligonucleotides are attached to a solid support;   (d) hybridizing the adaptor-ligated fragments to the oligonucleotides on the solid support so that 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 extended immobilized polynucleotides, thereby providing an array of amplified genomic fragments of different sequences present in different discreet locations of the array; and   (f) analyzing at least one of the amplified genomic fragments.   
     
     
         163 . The method of  claim 162  wherein the step of fragmenting a genomic DNA sample comprises fragmentation with a restriction endonuclease. 
     
     
         164 . The method of  claim 162  wherein the array of oligonucleotides is an array of regularly situated areas on a solid support, wherein different oligonucleotides are immobilized by covalent linkage. 
     
     
         165 . The method of  claim 164  wherein each oligonucleotide comprises a common region and a variable region. 
     
     
         166 . The method of  claim 164  wherein the variable regions vary in sequence or length. 
     
     
         167 . The method of  claim 163  wherein the step of ligating an adaptor to the fragments restores a recognition site for the restriction endonuclease. 
     
     
         168 . The method of  claim 160  wherein the adaptor sequence is appended to both ends of the fragments. 
     
     
         169 . The method of  claim 160  wherein prior to amplifying the extended immobilized oligonucleotides, the solid support is washed to remove non-covalently bound materials from the solid support. 
     
     
         170 . The method of  claim 160  wherein said oligonucleotides are attached to the solid support at the 5′ ends of the oligonucleotides. 
     
     
         171 . A method of analyzing a plurality of different nucleic acid sequences in a complex nucleic acid sequence comprising:
 (a) fragmenting the complex nucleic acid sample to obtain a plurality of different sequence nucleic acid fragments;   (b) ligating a first adaptor sequence to the 5′ ends of the fragments and a second adaptor sequence to the 3′ ends of the fragments, to obtain a plurality of different sequence, adaptor-ligated fragments;   (c) hybridizing the adaptor-ligated fragments to an array of oligonucleotides attached to a solid support wherein the oligonucleotides are attached to the solid support at the 5′ end and have a free 3′ end, and wherein the oligonucleotides comprise a sequence that is complementary to the second adaptor sequence;   (d) extending the oligonucleotides with a polymerase using the adaptor-ligated fragments as template to obtain extended oligonucleotides that comprise at their 3′ ends the complement of the first adaptor sequence;   (e) amplifying the extended oligonucleotides to obtain a plurality of different nucleic acid sequences by hybridizing a primer to the extended oligonucleotides, wherein the primer is complementary to the complement of the first adaptor sequence and extending the primer to obtain a copy of said extended oligonucleotides; and amplifying the copy of the extended oligonucleotide; and   (f) analyzing the plurality of different nucleic acid sequences.   
     
     
         172 . The method of  claim 171  wherein step (e) comprises extending said primers in the presence of a labeled nucleotide. 
     
     
         173 . The method of  claim 172  wherein said labeled nucleotide is a dideoxynucleotide.