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.

We claim:  
     
         1 . A binary oligonucleotide array comprising an array of predetermined areas on a surface of a solid support, each area having therein, covalently linked to said surface, multiple copies of a binary oligonucleotide of a predetermined sequence, said binary oligonucleotide consisting of a constant nucleotide sequence adjacent to a variable nucleotide sequence, wherein the constant nucleotide sequence is the same for all oligonucleotides in the array.  
     
     
         2 . A binary array according to  claim 1  wherein the binary oligonucleotides consist of deoxyribonucleotides.  
     
     
         3 . A binary array according to  claim 1  wherein the binary oligonucleotides consist of ribonucleotides.  
     
     
         4 . A binary array according to  claim 1  wherein one or more of nucleotides of the binary oligonucleotides are modified.  
     
     
         5 . A binary array according to  claim 1  wherein one or more of the nucleotides of the binary oligonucleotides are non-standard.  
     
     
         6 . A binary array according to  claim 1  wherein the binary oligonucleotides are mixed.  
     
     
         7 . A comprehensive binary array according to  claim 1   
     
     
         8 . A comprehensive binary array according to  claim 7  wherein the binary oligonucleotides in each area have variable sequences of the same length.  
     
     
         9 . A 3′ binary array according to  claim 1 .  
     
     
         10 . A 5′ binary array according to  claim 1 .  
     
     
         11 . A 3′ binary array according to  claim 9 , wherein each covalently linked binary oligonucleotide has its constant sequence adjacent to the 5′ end of its variable sequence.  
     
     
         12 . A 5′ binary array according to  claim 10 , wherein each covalently linked binary oligonucleotide has its constant sequence adjacent to the 3′ end of its variable sequence.  
     
     
         13 . A binary array according to  claim 2  wherein all or part of the constant nucleotide sequence is complementary to a predetermined restriction recognition sequence.  
     
     
         14 . A binary array according to  claim 1  having an oligonucleotide hybridized to all or part of the constant sequence which is ligatable to the terminus of an adjacent nucleic acid hybridized to the oligonucleotide.  
     
     
         15 . In an oligonucleotide array having variable-sequence oligonucleotides immobilized in a predetermined pattern of areas on a solid support, the improvement comprising including in said oligonucleotides a constant sequence of predetermined length.  
     
     
         16 . A sectioned binary array according to  claim 1 .  
     
     
         17 . A comprehensive sectioned binary array according to  claim 16 .  
     
     
         18 . A 3′ binary oligonucleotide array according to  claim 17 , wherein each covalently linked binary oligonucleotide has its variable sequence adjacent to the 5′ end of its constant sequence.  
     
     
         19 . A 5′ binary oligonucleotide array according to  claim 17 , wherein each covalently linked binary oligonucleotide has its variable sequence adjacent to the 3′ end of its constant sequence.  
     
     
         20 . A binary oligonucleotide array according to  claim 1 , wherein said constant nucleotide sequence comprises one or more functional sequences selected from the group consisting of a nucleic acid polymerase priming region, an RNA polymerase promoter region, and a restriction endonuclease recognition site.  
     
     
         21 . A binary oligonucleotide array according to  claim 20 , wherein said functional sequence is a priming region.  
     
     
         22 . A binary oligonucleotide array according to  claim 1 , wherein each binary oligonucleotide is covalently linked to said surface through a long polymer chain.  
     
     
         23 . A binary oligonucleotide according to  claim 2 , wherein said deoxyribonucleotides comprise at least one modified nucleotide.  
     
     
         24 . A sectioned oligonucleotide array comprising an array of predetermined areas on a surface of a solid support, each area having therein, covalently linked to said surface multiple copies of an oligonucleotide, wherein said areas are physically separated from one another into sections, such that nucleic acids in an aqueous solution generated in one section cannot migrate to another section.  
     
     
         25 . A sectioned oligonucleotide array according to  claim 24  further comprising a lattice attached to said surface.  
     
     
         26 . A sectioned oligonucleotide array according to  claim 25 , wherein said lattice is removably attached to said surface.  
     
     
         27 . A sectioned oligonucleotide array according to  claim 25 , further comprising a cover removably attachable to said lattice.  
     
     
         28 . A sectioned oligonucleotide array according to  claim 24 , wherein said sections comprise wells in said solid support.  
     
     
         29 . A sectioned oligonucleotide array according to  claim 28 , further comprising a cover removably attachable to said solid support.  
     
     
         30 . A sectioned oligonucleotide array according to  claim 24 , comprising a gel which physically separates said areas by preventing nucleic acids in an aqueous solution placed in one area from migrating to another area.  
     
     
         31 . A sectioned oligonucleotide array according to  claim 24 , wherein said sections are mechanically separated from one another.  
     
     
         32 . A sectioned oligonucleotide array according to  claim 27 , wherein said cover comprises a replica array.  
     
     
         33 . A sectioned oligonucleotide array according to  claim 29 , wherein said cover comprises a replica array.  
     
     
         34 . A sectioned array according to  claim 24  wherein all of the oligonucleotides in individual areas are of the same sequence.  
     
     
         35 . A sectioned array according to  claim 24  wherein not all oligonucleotides in each area are of the same sequence.  
     
     
         36 . A method of sorting a mixture of nucleic acid strands comprising the steps of: 
 a) providing a solution containing a mixture of nucleic acid strands in single-stranded form and    b) contacting said solution to a first binary oligonucleotide array of predetermined areas on a surface of a solid support, each area having therein, covalently linked to said surface, copies of a binary oligonucleotide, said binary oligonucleotide consisting of a constant nucleotide sequence adjacent to a variable nucleotide sequence, wherein the constant nucleotide sequence is the same for all oligonucleotides in the array, wherein said step of contacting is carried out under conditions promoting perfect hybridization of said strands to said binary oligonucleotides.    
     
     
         37 . A method according to  claim 36  wherein said array is comprehensive.  
     
     
         38 . A method according to  claim 36  wherein said array is a 3′ array.  
     
     
         39 . A method according to  claim 36  wherein said binary oligonucleotides are complementary to internal sequences that possibly occur in the strands in said mixture.  
     
     
         40 . A method according to  claim 39  wherein said array is comprehensive.  
     
     
         41 . A method according to  claim 36  wherein said array is a sectioned array, further comprising the step of amplifying strands hybridized in at least some of said areas to produce copies of said hybridized strands.  
     
     
         42 . A method according to  claim 39  wherein said step of providing comprises digesting genomic DNA with a restriction endonuclease to create DNA fragments; 
 (a) modifying said fragments by adding a first constant sequence to their strands' 3′ termini and a second constant sequence to their strands' 5′ termini to create priming regions including restored restriction sites; and  
 (b) denaturing the modified fragments to form a mixture of single nucleic acid strands.  
 
     
     
         43 . A method according to  claim 42  wherein said array is a sectioned, comprehensive array, further comprising the step of amplifying strands hybridized in said areas by symmetric PCR.  
     
     
         44 . A method according to  claim 42  further comprising the step of amplifying said mixture of single nucleic acid strands by asymmetric PCR.  
     
     
         45 . A method according to  claim 36  wherein said binary oligonucleotides or portions thereof are complementary to terminal sequences that possibly occur in one end of the strands in said mixture and that are substantially non-complementary to internal sequences in the strands in said mixture.  
     
     
         46 . A method according to  claim 45  wherein said array is a sectioned array, further comprising the step of amplifying strands hybridized in at least some of said areas to produce amplified copies of said single nucleic acid strands.  
     
     
         47 . A method according to  claim 46  wherein said array is a comprehensive array.  
     
     
         48 . A method according to  claim 46  wherein said array is a 3′ array.  
     
     
         49 . A method according to  claim 45  wherein said step of providing comprises digesting genomic DNA with a restriction endonuclease to create DNA fragments, modifying said fragments by adding a first constant sequence to their strands' 3′ termini to create priming regions including restored restriction sites, and denaturing the modified fragments into a mixture of single nucleic acid strands.  
     
     
         50 . A method according to  claim 45  wherein said step of providing comprises digesting genomic DNA with a restriction endonuclease to create DNA fragments; 
 (a) modifying said fragments by adding a first constant segment to one of their strands' 3′ and 5′ termini to create priming regions including restored restriction sites; and  
 (b) denaturing the modified fragments into a mixture of denatured nucleic acid strands each having a priming region only at one end.  
 
     
     
         51 . A method according to  claim 50  wherein said first binary sorting array is a 3′ array.  
     
     
         52 . A method according to  claim 51  further comprising the steps of 
 (a) generating an immobilized copy of each strand hybridized to the array by incubation with a DNA polymerase using the immobilized oligonucleotide as a primer and a hybridized strand as a template; and  
 (b) washing to remove from the array all materials not covalently bound to the array.  
 
     
     
         53 . A method according to  claim 52 , wherein said step of modifying comprises adding a first constant sequence to their strands' 5′ termini and wherein said 3′ array contains binary oligonucleotides to which are hybridized masking oligonucleotides, further comprising the steps of 
 (a) ligating said masking oligonucleotides to denatured nucleic acid strands hybridized to said binary oligonucleotides such that their 3′ termini are immediately adjacent to one of said masking oligonucleotides, and  
 (b) washing under conditions such that only strands so ligated will remain.  
 
     
     
         54 . A method according to  claim 53  wherein said step of adding a first constant sequence includes ligation of a double-stranded oligodeoxyribonucleotide adaptor.  
     
     
         55 . A method according to  claim 53  wherein said step of adding a first constant sequence includes ligation of a single-stranded oligoribonucleotide.  
     
     
         56 . A method according to  claim 52  wherein said step of modifying comprises adding a first constant sequence to their strands' 3′ termini.  
     
     
         57 . A method according to  claim 56  wherein said first constant sequence is a homopolynucleotide tail added by extension of the strands' 3′ termini by enzymatic extension.  
     
     
         58 . A method according to  claim 56  further comprising the step of adding a second constant sequence to the 3′ termini of the immobilized copies.  
     
     
         59 . A method according to  claim 58  wherein said second constant sequence is a homopolynucleotide tail added by extension of said immobilized copies' 3′ termini by enzymatic extension.  
     
     
         60 . A method according to  claim 52  wherein said first binary oligonucleotide array is a sectioned array, further comprising the step of amplifying said washed, immobilized copies to produce amplified copies.  
     
     
         61 . A method according to  claim 60  wherein said step of amplifying comprises PCR.  
     
     
         62 . A method according to  claim 60  wherein said first binary oligonucleotide array is a comprehensive array.  
     
     
         63 . A method according to  claim 60  further comprising contacting said amplified copies from at least one area of said 3′ array to a second binary oligonucleotide array containing immobilized binary oligonucleotides whose constant sequence is identical or complementary to the 3′ terminus of the immobilized copies.  
     
     
         64 . A method according to  claim 46  further comprising contacting said amplified copies from at least one area of said first binary oligonucleotide array to a second binary oligonucleotide array containing immobilized binary oligonucleotides that are complementary to terminal sequences that possibly occur in either the other ends of said denatured nucleic acid strands or the complements of said other ends, and that are not complementary to internal sequences in the strands in said mixture or their complements.  
     
     
         65 . A method according to  claim 45  wherein said step of providing comprises digesting genomic DNA with a restriction endonuclease to create DNA fragments, and denaturing said fragments into a mixture of denatured nucleic acid strands.  
     
     
         66 . A method according to  claim 65  wherein said first binary oligonucleotide array is a 3′ array containing binary oligonucleotides to which are hybridized masking oligonucleotides, further comprising the steps of ligating said masking oligonucleotides to denatured nucleic acid strands hybridized to said binary oligonucleotides such that their 3′ termini are immediately adjacent to one of said masking oligonucleotides, washing under conditions such that only strands so ligated will remain, and generating an immobilized copy of each ligated strand by incubation with a DNA polymerase.  
     
     
         67 . A method according to  claim 66  further comprising the steps of adding a constant sequence to the 5′ termini of the hybridized strands by ligation of a single-stranded oligoribonucleotide; incubating with a DNA polymerase to extend the immobilized copies; washing to remove from the array all materials not covalently bound to the array; and amplifying said washed, immobilized copies to produce amplified copies.  
     
     
         68 . A method according to  claim 67  wherein said step of amplifying comprises PCR.  
     
     
         69 . A method according to  claim 67  wherein said first sorting array is a comprehensive array.  
     
     
         70 . A method according to  claim 67  further comprising contacting said amplified copies from at least one area of said 3′ array to a second terminal binary array containing immobilized binary oligonucleotides whose constant sequence is identical or complementary to the 3′ terminus of said immobilized copies.  
     
     
         71 . A method according to  claim 51  further comprising the steps of adding a constant sequence to the 3′ termini of the immobilized copies by enzymatic extension thereof; washing to remove from the array all materials not covalently bound to the array; and amplifying said washed, immobilized copies to produce amplified copies.  
     
     
         72 . A method according to  claim 71  wherein said step of amplifying comprises PCR.  
     
     
         73 . A method according to  claim 71  wherein said first sorting array is a comprehensive array.  
     
     
         74 . A method according to  claim 71  further comprising contacting said amplified copies from at least one area of said 3′ array to a second terminal binary array containing immobilized binary oligonucleotides whose constant sequence is identical or complementary to the 3′ terminus of said immobilized copies.  
     
     
         75 . A method according to  claim 45  wheein said step of providing comprises digesting genomic DNA with a site-specific cleaving agent to create DNA fragments.  
     
     
         76 . A method according to  claim 75  wherein said agent is an endonuclease.  
     
     
         77 . A method according to  claim 75  wherein said agent is a chemical agent.  
     
     
         78 . A method according to  claim 45  wherein said nucleic acid strands are cDNA strands.  
     
     
         79 . A method according to  claim 45  wherein said nucleic acid strands are RNA strands.  
     
     
         80 . A method according to  claim 79  wherein said RNA strands are eukaryotic mRNA strands, and wherein said step of providing comprises removing 5′-cap structures.  
     
     
         81 . A method according to  claim 79  wherein said RNA strands lack a poly(A) tail.  
     
     
         82 . A method according to  claim 45  wherein said step of providing comprises digesting genomic DNA with a restriction endonuclease to create DNA fragments; 
 (a) modifying said fragments by adding a first constant sequence to their strands' 3′ termini and a second constant sequence to their strands' 5′ termini to create priming regions including restored restriction sites; and  
 (b) denaturing the modified fragments into a mixture of single nucleic acid strands.  
 
     
     
         83 . A method according to  claim 82  wherein the 3′ priming regions are complementary to the 5′ priming regions.  
     
     
         84 . A method according to  claim 83  wherein said array is a 3′ array, further comprising the steps of 
 (a) generating an immobilized copy of each strand hybridized to the array by incubation with a DNA polymerase; and  
 (b) washing to remove from the array all materials not covalently bound to the array.  
 
     
     
         85 . A method according to  claim 84  wherein said array is a sectioned array, further comprising the step of amplifying strands hybridized in at least some areas by PCR to produce amplified copies of each said immobilized copy.  
     
     
         86 . A method according to  claim 85  wherein said array is a comprehensive array.  
     
     
         87 . A method according to  claim 83  wherein addition of said first constant sequence and said second constant sequence includes ligation of a double-stranded oligodeoxyribonucleotide adaptor to the strands' 5′ termini.  
     
     
         88 . A method according to  claim 83  wherein addition of said first constant sequence and said second constant sequence includes ligation of a single-stranded oligonucleotide to the strands' 5′ termini.  
     
     
         89 . A method according to  claim 83  wherein addition of said first constant sequence and said second constant sequence includes enzymatic extension of the strands' 3′ termini by the synthesis of a homopolynucleotide tail.  
     
     
         90 . A method according to  claim 85  further comprising contacting said amplified copies from at least one areas of said 3′ array to a second binary array under conditions promoting hybridization of said amplified copies to the binary oligonucleotides in said second array.  
     
     
         91 . A method according to  claim 90  wherein said amplified copies are produced by symmetric PCR and wherein said second array is a 3′ array.  
     
     
         92 . A method according to  claim 90  wherein said first array and said second array are comprehensive.  
     
     
         93 . The product of a method according to  claim 84 .  
     
     
         94 . A method of sorting a mixture of nucleic acid strands comprising the steps of 
 a) providing a solution containing a mixture of nucleic acid strands in single stranded form, and    b) contacting said solution to an oligonucleotide array of predetermined areas on a surface of a solid support, each area having therein copies of an immobilized oligonucleotide, the nucleotide sequence of immobilized oligonucleotides in separate areas being different, wherein said contacting is performed under conditions that promote the formation of perfect hybrids.    
     
     
         95 . A method according to  claim 94  wherein said array is comprehensive.  
     
     
         96 . A method according to  claim 94  wherein the array is sectioned.  
     
     
         97 . A method according to  claim 94  wherein the immobilized oligonucleotides are between 6 and 30 nucleotides long.  
     
     
         98 . A method according to  claim 94  wherein the array is a 3′ array.  
     
     
         99 . A method according to  claim 94  wherein the array is a 5′ array.  
     
     
         100 . In a method wherein two nucleic acid strands are ligated to each other in order to form a recombinant product, the improvement comprising hybridizing first nucleic acid strands to immobilized oligonucleotides in an oligonucleotide array prior to ligation to second nucleic acid strands, said oligonucleotide array comprising an array of predetermined areas on a surface of a solid support, each area having copies of an oligonucleotide immobilized thereon.  
     
     
         101 . A method according to  claim 100  wherein the first nucleic acid strands have different nucleotide sequences in each area of the array.  
     
     
         102 . A method according to  claim 100  wherein the second nucleic acid strands have different nucleotide sequences in each area of the array.  
     
     
         103 . A method according to  claim 100  wherein the array is a comprehensive array.  
     
     
         104 . A method acording to  claim 100  wherein the oligonucleotides immobilized in each area are of the same length.  
     
     
         105 . A method according to  claim 100  wherein the oligonucleotides consist of the group consisting of deoxyribonucleotides, ribonucleotides, mixed deoxyribonucleotides and ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, and non-standard nucleotides.  
     
     
         106 . A method according to  claim 100  wherein the second nucleic acid strands are not also hybridized to the immobilized oligonucleotides.  
     
     
         107 . A method according to  claim 106  wherein the second nucleic acid strands are strands of double stranded nucleic acids.  
     
     
         108 . A method according to  claim 107  wherein the set of double stranded nucleic acids has one end adapted for ligation to blunt ends formed by hybridization of the first set of nucleic acids to the immobilized oligonucleotides.  
     
     
         109 . A method according to  claim 108  wherein nonligating termini of the first nucleic acid strands and the double stranded nucleic acids contain priming regions for amplification.  
     
     
         110 . A method according to  claim 107  wherein following ligation of the first nucleic acids to the second nucleic acids, polymerase chain reaction amplification is carried out.  
     
     
         111 . A method according to  claim 108  wherein the double stranded nucleic acids are ligated to the immobilized oligonucleotide using RNA ligase prior to ligation of the first nucleic acid strands and the second nucleic acid strands.  
     
     
         112 . A method according to  claim 107  wherein the second set of nucleic acids is the same in every area of array.  
     
     
         113 . A method according to  claim 107  wherein the first nucleic strands are hybridized to the immobilized oligonucleotides while contained in one or more mixtures of different strands, said different strands having different terminal sequences from corresponding termini to be ligated of the first nucleic acid strands.  
     
     
         114 . A method according to  claim 100  wherein both the first nucleic acid strands and the second nucleic acid strands are hybridized to the immobilized oligonucleotides in the array prior to ligation.  
     
     
         115 . A method according to  claim 114  wherein both the first and second nucleic acid strands contain priming regions at their non-ligating termini.  
     
     
         116 . A method according to  claim 115  wherein the first and second nucleic acid strands are amplified in a polymerase chain reaction following ligation.  
     
     
         117 . A method according to  claim 114  wherein both the first, and second nucleic acids are, prior to hybridization to the immobilized oligonucleotides, contained in mixtures of nucleic acids having different terminal sequences from the corresponding termini to be ligated of the first nucleic acid strands and the second nucleic acid strands.  
     
     
         118 . A method for introducing site directed mutations into a nucleic acid strand in parallel on an oligonucleotide array using partials, said partials corresponding to regions of the nucleic acid strand adjacent to the location of site directed mutations to be introduced, comprising the steps: 
 (a) separately ligating said partials to free termini of preselected immobilized oligonucleotides in an oligonucleotide array to obtain mutated partials, said oligonucleotide array comprising an array of predetermined areas on the surface of a solid support, each area having therein a preselected immobilized oligonucleotide, said preselected oligonucleotide in each area having a sequence adapted to introduce a sequence containing the site directed mutation to a partial added to that area; and    (b) utilizing the mutated partial to generate a nucleic acid containing the site directed mutation.    
     
     
         119 . A method according to  claim 118  wherein step b is accomplished by 
 (a) hybridizing a complementary copy of the mutated partial to a template having the complementary sequence of the terminal portion of the nucleic acid strand which is not contained in the partial; and  
 (b) carrying out a polymerase reaction, a ligation reaction or both a polymerase reaction and ligation reaction to join the remaining region of the nucleic acid strand to the mutated partial.  
 
     
     
         120 . A method for making immobilized partial copies of a nucleic acid strand on a 3′ or 5′ oligonucleotide array, comprising the step: 
 (a) hybridizing the strand to the array by an oligonucleotide segment contained in the strand, said array comprising predetermined areas on a surface of a solid support, each area having therein immobilized oligonucleotides consisting of a predetermined variable sequence, said hybridization taking place under conditions that promote the formation of perfect hybrids of the length of the immoblized oligonucleotide in each area, and  
 (b) where the strand is hybridized to a 3′ array, enzymatically extending the immobilized oligonucleotide using the hybridized strand as a template, and where the strand is hybridized to a 5′ array, hybridizing a primer to a priming region contained in a fixed end of the partial, then enzymatically extending the primer to form an extension product, then ligating the extension product to the immobilized oligonucleotide.  
 
     
     
         121 . A method according to  claim 120  wherein the strand is hybridized to a 3′ array, further comprising amplifying the immobilized partial copies using a primer or promoter complement appropriate to hybridize to a priming region or promoter sequence at the partial copy's fixed terminus, and an appropriate polymerase.  
     
     
         122 . A method according to  claim 120  wherein the oligonucleotide array is substantially comprehensive.  
     
     
         123 . A method according to  claim 122  wherein a substantially complete set of immobilized partial copies is generated on the array by 
 (a) hybridizing the strand to the array by substantially all oligonucleotides present in the strand;  
 (b) performing step (b) on all hybridized strands.  
 
     
     
         124 . A method according to  claim 122  wherein a substantially complete set of amplified partials is generated on a 3′ array by 
 (a) hybridizing the strand to the 3′ array by substantially all oligonucleotides present in the strand;  
 (b) performing step (b) on all hybridized strands; and  
 (c) amplifying substantially all immobilized partial copies by using a primer or promoter complement appropriate to hybridize to a priming region or promoter sequence at the partial copy's fixed terminus, and an appropriate polymerase.  
 
     
     
         125 . A method according to  claim 123 ,  124  wherein following step a unhybridized and imperfectly hybridized strand copies are removed.  
     
     
         126 . A method according to  claim 125  wherein the array is sectioned.  
     
     
         127 . A method according to  claim 126  wherein the strand is contained in a mixture of strands which are subjected to the same steps on the array.  
     
     
         128 . A method according to  claim 125  wherein the priming region or promoter is added to the 5′ terminus of the nucleic acid strand prior to hybridizing the strand to the array.  
     
     
         129 . A method according to  claim 126  further wherein the oligonucleotide content in an area of the array is surveyed.  
     
     
         130 . The product of a method according to  claim 120 .  
     
     
         131 . The product of a method according to  claim 122 .  
     
     
         132 . A method for sorting partials by their variable termini on a binary oligonucleotide array, which partials have been prepared by random chemical or enzymatic degration of one or more nucleic acid strands, said binary array comprising an array of predetermined areas on a surface of a solid support, each area having therein copies of a binary oligonucleotide of a predetermined sequence, said binary oligonucleotide consisting of a constant nucleotide sequence adjacent to a variable nucleotide sequence, said variable nucleotide sequence being at the free end of the binary oligonucleotides, said binary oligonucleotide also having a complementary masking oligonucleotide hybridized to all or a part of the constant nucleotide sequence, including the portion of the constant nucleotide sequence adjacent the variable nucleotide sequence, comprising the steps of: 
 (a) hybridizing the partials to the array by their termini under conditions that promote the formation of perfect hybrids; and  
 (b) ligating the termini of the partials to the masking oligonucleotide.  
 
     
     
         133 . A method for obtaining information for determining the sequence of a nucleic acid strand comprising 
 (a) generating a substantially complete set of partials of the nucleic acid strand; and    (b) for groups of partials, having the same terminal variable nucleotide sequence of predetermined length, separately determining the presence and sequence of all variable oligonucleotides of the predetermined length.    
     
     
         134 . In a method for surveying oligonucleotide content of a nucleic acid strand as part of a sequencing method wherein the strand is hybridized to a comprehensive oligonucleotide array, and the presence of hybridized strands in areas of the array is detected, the improvement comprising: 
 (a) preparing a substantially complete set of partials of the strand prior to surveying;    (b) sorting the partials by their variable ends on an oligonucleotide array, and    (c) separately surveying oligonucleotide content of each group of sorted partials.    
     
     
         135 . A method according to  claim 134  wherein the strand is in a mixture of strands which are subjected to the same steps.  
     
     
         136 . A method according to  claim 135  wherein the substantially complete set of partials is prepared by chemical or enzymatic degradation of the strands and the strands are sorted on a binary oligonucleotide array, said binary array comprising an array or predetermined areas on a surface of a solid support, each area having therein copies of an binary oligonucleotides of a predetermined sequence, said binary oligonucleotide consisting of a constant nucleotide sequence of predetermined length and nucleotide sequence adjacent to a variable nucleotide sequence.  
     
     
         137 . A method according to  claim 136  wherein said binary oligonucleotide array comprises a 3′ array, said immobilized oligonucleotides consisting of a constant sequence at the 5′ terminus of a variable sequence.  
     
     
         138 . A method according to  claim 135  further comprising 
 (a) preparing address sets containing a complete list of all oligonucleotides contained in a strand or strands in the mixture which share an address oligonucleotide for substantially every address in the oligonucleotide array on which the partials were sorted; and  
 (b) determining whether an address set is a strand set by examining whether the address set can be decomposed into other address sets.  
 
     
     
         139 . A method according to  claim 138  further comprising organizing the oligonucleotides in a strand set into sequence blocks composed of oligonucleotides that uniquely overlap each other, and ordering the blocks.  
     
     
         140 . A method of obtaining information to order a set of first fragments resulting from digestion of DNA with a first restriction endonuclease, the nucleotide sequence of said fragments having already been determined, comprising 
 (a) digesting the DNA with a second restriction endonuclease to generate a set of second fragments;    (b) denaturing the second set of fragments to form a mixture of single nucleic acid strands;    (c) sorting strands on a substantially comprehensive oligonucleotide array;    (d) amplifying the strands to generate both their direct and complementary copies;    (e) surveying the contents of individual areas of the array on a first binary survey array, said first binary survey array comprising an array of predetermined areas on a surface of a solid support, each area having therein, covalently linked to said surface, copies of a binary oligonucleotide, said binary oligonucleotide having a constant nucleotide sequence which contains a sequence complementary to the restriction recognition site of the first restriction endonuclease and adjacent to a variable sequence; and    (f) surveying the contents of individual areas of the array on a second binary survey array, said second binary survey array comprising an array of predetermined areas on a surface of a solid support, each area having therein, covalently linked to said surface, copies of a second binary oligonucleotide, said second binary oligonucleotide having a constant nucleotide sequence which contains a sequence complementary to the restriction recognition site of the second restriction endonuclease and adjacent to a variable sequence.    
     
     
         141 . A method according to  claim 140  wherein in step c strands are hybridized to an array selected from the group consisting of 
 (a) a first binary sorting array, said first binary sorting array comprising an array of immobilized oligonucleotides having a constant nucleotide sequence complementary to the restriction recognition site of the first restriction endonuclease, adjacent to a variable sequence of predetermined length, the immobilized oligonucleotides in an individual area of the first binary sorting array having the same sequence, and  
 (b) a second binary sorting array, said second binary sorting array comprising an array of immobilized oligonucleotides having a constant nucleotide sequence complementary to the restriction recognition site of the second restriction endonuclease, adjacent to a variable sequence of predetermined length, the immobilized oligonucleotides in an individual area of the second binary sorting array having the same sequence,  
 and wherein following hybridization unhybridized and imperfectly hybridized strands are removed.  
 
     
     
         142 . A method for obtaining information to allocate sequenced and ordered fragments from an original restriction digest of DNA from sister chromosomes to chromosomal linkage groups comprising 
 (a) preparing a partial on an oligonucleotide array from a restriction fragment from an alternate restriction digest of the DNA, which partial spans first and second allelic differences in neighboring pairs of sequenced fragments from the original restriction digest; and    (b) determining the presence of oligonocleotides containing the first and second allelic diffences in a partial which spans the first and second allelic differences.    
     
     
         143 . A method according to  claim 142  wherein 
 (a) in step b, the restriction fragment from the alternate restriction digest is hybridized to the oligonucleotide array by an oligonucleotide containing the first allelic difference; and  
 (b) the presence of an oligonucleotide containing the second allelic difference is determined by hybridizing the partial to a complementary second variable nucleotide sequence in an oligonucleotide array and then detecting the presence of the partial in the corresponding area of the oligonucleotide array.  
 
     
     
         144 . A method for surveying oligonucleotides in a nucleic acid strand comprising 
 (a) randomly degrading the strand into pieces, the average length of said pieces slightly exceeding the length of oligonucleotides surveyed;    (b) ligating the pieces to a ligating oligonucleotide complementary to at least a portion of a constant sequence of immobilized oligonucleotides in a binary array;    (c) hybridizing the pieces to the binary array, said binary array having immobilized oligonucleotides in an ordered array therein and consisting of a constant sequence adjacent to a variable sequence, the immobilized oligonucleotides in an individual area of the array having the same sequence; and    (d) detecting the hybrids formed.    
     
     
         145 . A method according to  claim 144  wherein the array is a 3′ array having the variable sequence at the 3′ termini of the immobilized oligonucleotides, further comprising, following step c, 
 (a) extending the immobilized oligonucleotides with a polymerase using hybridized pieces as templates.  
 
     
     
         146 . A method according to  claim 145  wherein the strand is a DNA strand resulting from a digest with a restriction endonuclease, and melting apart of the fragments obtained thereby or a partial obtained from said strand, and wherein the constant sequence contains the restriction endonuclease recognition site.  
     
     
         147 . A method according to  claim 146  wherein dideoxynucleotides are used as substrates during extension of the immobilized oligonucleotides using a DNA polymerase.  
     
     
         148 . In a primer dependent polymerase reaction for amplification of a nucleic acid in which a primer is hybridized to a template strand and extended by incubation with a primer dependent polymerase and nucleotide substrates to generate a complementary copy of the template strand; the improvement wherein: 
 the primer or a part thereof contains one or more primer nucleotides that are chemically different from nucleotide substrates incorporated in the complementary copy of the template during the amplification said chemical difference causing the primer to be cleavable without cleaving the part of the complementary copy generated during amplification.    
     
     
         149 . A method according to  claim 148  further wherein the primer is selectively cleaved without cleaving the part of the complementary copy generated during amplification.  
     
     
         150 . A method according to 149 wherein the primer or a part thereof contains one or more ribonucleotides triphosphates, and the substrates used for amplification are deoxyribonucleoside triphosphates and the primer is cleaved by a chemical or enzymatic reaction which cleaves nucleic strands immediately 3′ of ribonucleotides but not 3′ of deoxyribonucleotides.  
     
     
         151 . A method according to  claim 150  wherein the chemical reaction or enzymatic reaction is selected from the group consisting of 
 (a) alkaline hydrolysis;  
 (b) hydrolysis by a magnesium formamide mixture;  
 (c) ribonuclease digestion.  
 
     
     
         152 . A method according to  claim 150  wherein a ribonucleotide is present a the 3′ terminus of the primer.  
     
     
         153 . A method according to  claim 149  wherein said nucleotide substrates used for amplification are modified at their alpha phosphate groups so that resulting modified phosphodiester bonds in the complementary copy generated during amplification is resistant to cleavage by a nuclease, said nuclease being chosen to be incapable of cleaving said resulting modified phosphodiester bonds, further wherein one or more primer phosphodiester bonds are not modified to be resistant to said cleavage, and wherein said primer is cleaved by treatment with said nuclease.  
     
     
         154 . A method according to  claim 153  wherein said nucleotide substrates modified at their alpha phosphate groups are nucleoside alpha-thiophosphates.  
     
     
         155 . A method according to  claim 153  wherein the nucleotide substrates used for amplification are modified deoxyribonucleotides.  
     
     
         156 . An array of oligonucleotide arrays comprising a solid sheet having a surface and an array comprising a pattern of miniaturized oligonucleotide arrays on said surface, each miniaturized array comprising an array of predetermined areas on said surface, each area having therein, covalently linked to said surface, multiple copies of an oligonucleotide of a predetermined sequence.  
     
     
         157 . A method according to  claim 52  further comprising 
 (a) contacting at least one area of said array containing the immobilized copies with at least one oligonucleotide probe having a predetermined sequence, under conditions promoting hybridization of said at least one probe; and  
 (b) determining whether or not said at least one probe has hybridized to said at least one area.  
 
     
     
         158 . A method according to  claim 120  further comprising 
 (a) contacting at least one area of said array containing the immobilized partial copies with at least one oligonucleotide probe having a predetermined sequence, under conditions promoting hybridization of said at least one probe; and  
 (b) determining whether or not said at least one probe has hybridized to said at least one area.  
 
     
     
         159 . A method according to  claim 133 , wherein determining the presence and sequence of all variable oligonucleotides comprises 
 (a) contacting said substantially complete set of partials with a substantially comprehensive set of oligonucleotide probes, each of a predetermined length, under conditions promoting hybridization of said probes; and    (b) determining to which partials each said probe has hybridized.