Methods and devices for measuring differential gene expression

Abstract

This invention includes methods for identifying nucleic acids in a sample of nucleic acids by observing sequence sets present in the nucleic acids of the sample and then identifying those sequences in a nucleic acid sequence database having the sequence sets observed. In a preferred embodiment, a sequence set consists of two primary subsequences and an additional subsequence having determined mutual relationships. The methods include those for observing the sequence sets and those for performing sequence database searches. This invention also includes devices for recognizing in parallel the additional subsequences in a sample of as well as methods for the use of these devices. In a preferred embodiment, the devices include probes bound to a planar surface that recognize additional subsequence by hybridization, and the methods of use include features to improve the specificity and reproducibility of this hybridization.

What is claimed is:  
     
         1 . A method for identifying and quantifying nucleic acids in a sample of nucleic acids comprising: 
 (a) observing subsequence sets present in said sample of nucleic acids, wherein a subsequence set comprises at least two nucleotide subsequences in a non-adjacent arrangement and said subsequence set is observed in said sample if a nucleic acid in said sample includes said two nucleotide subsequences in a non-adjacent arrangement; and    (b) searching a database of nucleic acid sequences in order to locate database sequences having said observed subsequence sets or to determine that no such database sequences exist, said database of nucleic acid sequences comprising nucleic acid sequences that might be present in said sample;    thereby identifying said located database sequences as sequences of nucleic acids present in said sample.    
     
     
         2 . The method of  claim 1  wherein said subsequence set comprises a first primary nucleotide subsequence, a second primary nucleotide subsequence, and an additional nucleotide subsequence, and wherein said additional nucleotide subsequence is spaced apart from said first primary nucleotide subsequence by a distance of zero or more nucleotides which is the same in all subsequence sets and said second primary nucleotide subsequence is not adjacent to said additional nucleotide subsequence.  
     
     
         3 . The method of  claim 2  wherein said fixed distance is zero nucleotides, whereby said subsequence set comprises two non-adjacent subsequences.  
     
     
         4 . The method of  claim 2  wherein the length of said first primary nucleotide subsequence is at least six nucleotides, the length of said second primary nucleotide subsequence is at least six nucleotides, and the length of said additional nucleotide subsequence is at least four nucleotides.  
     
     
         5 . The method of  claim 2  wherein the length of said first primary nucleotide subsequence is six nucleotides, the length of said second primary nucleotide subsequence is six nucleotides, and the length of said additional nucleotide subsequence is at least six nucleotides.  
     
     
         6 . The method of  claim 2  wherein the length of said first primary nucleotide sequence, the length of said second primary nucleotide sequence, and the length of said additional nucleotide sequence are chosen such that on average a particular subsequence set occurs in less than approximately 10 sequences in said sequence database.  
     
     
         7 . The method of  claim 2  wherein the length of said first primary nucleotide sequence, the length of said second primary nucleotide sequence, and the length of said additional nucleotide sequence are chosen such that on average a particular subsequence set occurs in less than approximately sequences in said sequence database.  
     
     
         8 . The method of  claim 2  wherein said step of observing comprises the steps of: 
 (a) providing at least one subsample of first nucleic acid fragments, said first nucleic acid fragments in said subsample being derived from those nucleic acids in said sample in which said first and said second primary nucleotide subsequences have selected sequences; and  
 (b) determining the sequence of said additional nucleotide subsequence in each said first nucleic acid fragment of said subsample.  
 
     
     
         9 . The method of  claim 8  wherein said providing step provides a plurality of subsamples, each subsample having different selected sequences for said first or said second primary nucleotide subsequences from other subsamples.  
     
     
         10 . The method of  claim 8  wherein the length of said first primary nucleotide sequence, the length of said second primary nucleotide sequence, and the length of said additional nucleotide sequence are chosen such that on average the number of species of first nucleic acid fragments in said subsample is less than the number of possible sequences for said additional nucleotide subsequences.  
     
     
         11 . The method of  claim 8  wherein said first and said second primary nucleotide subsequences are sequences of recognition sites of restriction endonucleases of a type that cuts nucleic acids within their recognition sites, and wherein said providing step comprises digesting said sample with restriction endonucleases whose recognition sites have the sequences selected for said first and second primary nucleotide subsequences.  
     
     
         12 . The method of  claim 11  wherein said providing step further comprises, after said digesting, the steps of (i) hybridizing digested nucleic acids with adapter nucleic acids, said adapter nucleic acids being partially double-stranded, each said adapter nucleic acid being complementary to an end of a nucleic acid digested by one of said restriction endonucleases, and (ii) ligating said hybridized nucleic acids and adapter nucleic acids.  
     
     
         13 . The method of  claim 8  wherein said determining step comprises: 
 (a) producing second nucleic acid fragments from said first nucleic acid fragments of said subsample, wherein said second nucleic acid fragments have a single-stranded terminal nucleotide subsequence, and wherein said additional nucleotide subsequence comprises said single-stranded terminal nucleotide subsequence;  
 (b) hybridizing a plurality of species of probe molecules with said second nucleic acid fragments, probe molecules of each of said species of probe molecules capable of hybridizing with said second nucleic acid fragments having a particular sequence for said additional nucleotide subsequence; and  
 (c) detecting which of said species of probe molecules has hybridized with said second nucleic acid fragments;  
 whereby the sequences of said additional nucleotide sequences are determined.  
 
     
     
         14 . The method of  claim 13  wherein said species of probe molecules are fixed on a surface in an arrangement such that each species of probe molecule occupies a discrete observational region of said surface separate from regions occupied by other species of probe molecules.  
     
     
         15 . The method of  claim 13  further comprising, before said detecting step, a step of washing said probe molecules hybridized with said second nucleic acid fragments at a stringency to remove mis-hybridized or non-specifically bound second nucleic acid fragments.  
     
     
         16 . The method of  claim 13  wherein said second fragments are labeled with a detectable moiety.  
     
     
         17 . The method of  claim 16  wherein said detectable moiety is a fluorescent moiety or a moiety recognizable by an antibody conjugated to a detectable moiety.  
     
     
         18 . The method of  claim 13  wherein said producing step comprises digesting said fragments in said subsample with a restriction endonuclease of a type that cuts nucleic acids outside of its recognition site.  
     
     
         19 . The method of  claim 13  wherein said probe molecules comprise a peptido-nucleic acid subsequence, such that for each species of said probe molecule the sequence of said peptido-nucleic acid subsequence is complementary to the sequence of said additional subsequence hybridizable to said species of probe molecules.  
     
     
         20 . The method of  claim 13  wherein said probe molecules comprise a nucleotide sequence, which in turn comprises a hybridization region nucleotide subsequence and a core nucleotide subsequence, the sequence of said hybridization region nucleotide subsequence being complementary to the sequence of said additional subsequence hybridizable to said species of probe molecules, said core nucleotide subsequence being adjacent to said hybridization region nucleotide subsequence, and wherein said step of hybridizing comprises: 
 (a) hybridizing a plurality of species of probe molecules with said second nucleic acid fragments and with stacking oligomers to form a hybridization structure, the sequence of said stacking oligomers being complementary to a hybridizable portion of the sequence of said core nucleotide subsequence of said probe molecules, said hybridizable portion being adjacent to said hybridization region nucleotide subsequence; and  
 (b) ligating nicks in said hybridization structure.  
 
     
     
         21 . The method of  claim 20  wherein said additional nucleotide subsequence consists of said single-stranded terminal nucleotide subsequence of said second nucleic acid fragments.  
     
     
         22 . The method of  claim 20  wherein said additional nucleotide subsequence comprises said single-stranded terminal nucleotide subsequence of said second nucleic acid fragments and a double-stranded nucleotide subsequence of said second nucleic acid fragments, said double-stranded terminal nucleotide subsequence being adjacent to said single-stranded nucleotide subsequence.  
     
     
         23 . The method of  claim 20  wherein said stacking oligomers are labeled with a detectable moiety.  
     
     
         24 . The method of  claim 23  wherein said detectable moiety comprises a fluorescent energy transfer dye.  
     
     
         25 . The method of  claim 24  wherein said fluorescent energy transfer dye comprises a donor moiety and an acceptor moiety, and wherein one of said donor or said acceptor moieties is not conjugated to said stacking oligomer.  
     
     
         26 . The method of  claim 20  wherein said second nucleic acid fragments are labeled with a detectable moiety.  
     
     
         27 . The method of  claim 26  wherein said detectable moiety comprises a fluorescent energy transfer dye, wherein said fluorescent energy transfer dye comprises a donor moiety and an acceptor moiety, and wherein one of said donor or said acceptor moieties is not conjugated to said second nucleic acid fragments.  
     
     
         28 . The method of  claim 20  wherein said hybridizing and said ligating steps are (i) performed in conditions such that the concentration of said second nucleic acid fragments is less than concentrations of said probe molecules, and (ii) performed for a time less than that after which concentrations of second nucleic acid fragments become depleted.  
     
     
         29 . The method of  claim 13  wherein said detecting step additionally detects the amount of said second nucleic acid fragments hybridized with said species of probe molecule.  
     
     
         30 . The method of  claim 8  further comprising, after said determining step, a step of amplifying one or more first nucleic acid fragments in said subsample by contacting said subsample with a nucleic acid polymerase and one or more primers, at least one primer being a phasing primer which comprises a subsequence with the same sequence as one of said determined additional subsequences.  
     
     
         31 . The method of  claim 30  further comprising, after said amplifying step, a step of sequencing at least a portion of one or more nucleic acid products of said amplifying step.  
     
     
         32 . The method of  claim 31  wherein said determined sequences are compared with said located database sequences that contain a sequence set comprising said selected first primary nucleotide sequence, said selected first primary nucleotide sequences, and said determined additional subsequence.  
     
     
         33 . The method of  claim 32  further comprising, after said amplifying and searching steps, storing the results of said amplifying in a permanent storage.  
     
     
         34 . The method of  claim 1  wherein said searching step further comprises examining individually and sequentially each sequence in the sequence database for the presence of a sequence set.  
     
     
         35 . The method of  claim 1  wherein said searching step comprises representing a sequence set as a regular expression in order to search sequences in the sequence database.  
     
     
         36 . The method of  claim 1  further comprising, prior to said searching step, a step of constructing an index of subsequences present in the sequences of said sequence database, and wherein said searching step consults said index of subsequences.  
     
     
         37 . The method of  claim 1  further comprising, after said searching step, a step of storing said located sequences in a permanent computer-readable storage.  
     
     
         38 . The method of  claim 37  where said step of storing stores along with said located sequences additional information describing said sample of nucleic acids.  
     
     
         39 . The method of  claim 37  wherein said step of observing further observes the amount of nucleic acids in said sample having said observed subsequence sets, and wherein said step of storing stores along with said located sequences said observed amount.  
     
     
         40 . A computer readable storage medium produced according to the method of  claim 37 .  
     
     
         41 . A method for identifying nucleic acids in a sample of nucleic acids comprising: 
 (a) providing at least one subsample of first nucleic acid fragments, said first nucleic acid fragments in said subsample being derived from those nucleic acids in said sample which have selected sequences for a first primary nucleotide subsequence and a second primary nucleotide subsequence, wherein said second primary nucleotide subsequence is not adjacent to said first primary nucleotide subsequence;    (b) determining the sequence of an additional subsequence in said first nucleic acid fragments of said subsample, wherein said additional subsequence is spaced apart from said first primary nucleotide subsequence by a fixed displacement of zero of more nucleotides; and    (c) amplifying first nucleic acid fragments in said subsample by contacting said subsample with a nucleic acid polymerase and one or more primers, at least one primer being a phasing primer which comprises a subsequence with the same sequence as one of said determined additional subsequences.    
     
     
         42 . The method of  claim 41  further comprising, after said amplifying step, a step of sequencing at least a portion of one or more nucleic acid products of said amplifying step.  
     
     
         43 . The method of  claim 41  wherein said first and said second primary nucleotide subsequences are sequences of recognition sites of restriction endonucleases of a type that cuts nucleic acids within their recognition sites, and wherein said providing step comprises digesting said sample with restriction endonucleases whose recognition sites have the sequences selected for said first and second primary nucleotide subsequences.  
     
     
         44 . The method of  claim 43  wherein said providing step further comprises, after said digesting, the steps of (i) hybridizing digested nucleic acids with adapter nucleic acids, said adapter nucleic acids being partially double-stranded and each said adapted nucleic acid being complementary to an end of a nucleic acid digested by one of said restriction endonucleases, and (ii) ligating said hybridized nucleic acids and adapter nucleic acids.  
     
     
         45 . The method according to  claim 44  wherein said adapters comprise a common adapter subsequence, and wherein said primers comprise hybridizable portions of said common adapter subsequence.  
     
     
         46 . The method of  claim 41  wherein said determining step comprises: 
 (a) producing second nucleic acid fragments from said first nucleic acid fragments of said subsample, wherein said second nucleic acid fragments have a single-stranded terminal nucleotide subsequence, said additional nucleotide subsequence comprising said single-stranded terminal nucleotide subsequence;  
 (b) hybridizing a plurality of species of probe molecules with said second nucleic acid fragments, each of said species of probe molecules capable of hybridizing with said second nucleic acid fragments having a selected sequence for said additional nucleotide subsequence; and  
 (c) detecting which of said species of probe molecules has hybridized with said second nucleic acid fragments. whereby the sequences of said additional nucleotide sequences are determined.  
 
     
     
         47 . A method for identifying and quantifying nucleic acids in a sample of nucleic acids comprising: 
 (a) providing at least one subsample of first nucleic acid fragments, said first nucleic acid fragments in said subsample being derived from those nucleic acids in said sample in which a first primary nucleotide subsequence and a second primary nucleotide subsequence have selected sequences,    wherein said first and said second primary nucleotide subsequences are not contiguous in said nucleic acids;    (b) producing second nucleic acid fragments having a single-stranded terminal nucleotide subsequence from said subsample of first nucleic acid fragments;    (c) determining a sequence for an additional nucleotide subsequence of said second nucleic acid fragments, said additional nucleotide subsequence comprising said single-stranded terminal nucleotide subsequence, and wherein said single-stranded nucleotide subsequence is spaced apart from said first primary nucleotide subsequence by a distance of zero or more nucleotides which is the same in all second nucleic acid fragments, said determining by: 
 (i) hybridizing a plurality of species of probe molecules with said second nucleic acid fragments, each of said species of probe molecules capable of hybridizing with said second nucleic acid fragments having a particular sequence for said additional nucleotide subsequence, and  
 (ii) detecting which of said species of probe molecules has hybridized with said second nucleic acid fragments, and the amount of said second nucleic acid fragments hybridized with said species of probe molecule;  
   (d) searching a database of nucleic acid sequences in order to locate database sequences having said selected first primary subsequence, said selected second primary subsequence, and said determined additional subsequence or to determine that no such database sequences exist, said database of nucleic acid sequences comprising nucleic acid sequences that might be present in said sample;    thereby identifying said located database sequences as sequences of nucleic acids present in said sample.    
     
     
         48 . The method of  claim 47  wherein said probe molecules comprise a nucleotide sequence, which in turn comprises a hybridization region nucleotide subsequence and a core nucleotide subsequence, the sequence of said hybridization region nucleotide subsequence being complementary to the sequence of said additional subsequence hybridizable to said species of probe molecules, said core nucleotide subsequence being adjacent to said hybridization region nucleotide subsequence, and wherein said step of hybridizing comprises: 
 (a) hybridizing a plurality of species of probe molecules with said second nucleic acid fragments and with stacking oligomers to form a hybridization structure, the sequence of said stacking oligomers being complementary to a hybridizable portion of the sequence of said core nucleotide subsequence of said probe molecules, said hybridizable portion being adjacent to said hybridization region nucleotide subsequence; and  
 (b) ligating nicks in said hybridization structure.  
 
     
     
         49 . The method of  claim 47  wherein said additional nucleotide subsequence consists of said single-stranded terminal nucleotide subsequence of said second nucleic acid fragments.  
     
     
         50 . The method of  claim 47  wherein said additional nucleotide subsequence comprises said single-stranded terminal nucleotide subsequence of said second nucleic acid fragments and a double-stranded nucleotide subsequence of said second nucleic acid fragments, said double-stranded terminal nucleotide subsequence being adjacent to said single-stranded nucleotide subsequence.  
     
     
         51 . A detection array for recognizing terminal subsequences of target nucleic acids, said array comprising: 
 (a) one or more surfaces;    (b) a plurality of discrete observational cells arranged on said surfaces in which are bound probe molecules, each probe molecule being a member of one of a plurality of species of probe molecules, wherein each discrete observational cell has bound probe molecules of at most one species, and wherein said probe molecules comprise: 
 (i) a hybridization region, wherein said hybridization region of said probe molecules of one species of probe molecule are capable of hybridizing with said terminal subsequences of said target nucleic acids having a single nucleotide sequence,  
 (ii) a core region adjacent to and conjugated with said hybridization region, and  
 (iii) an attachment means for binding said hybridization region and said core region to said surfaces; and  
   (c) a plurality of discrete error-checking cells to which are bound probe molecules, wherein to each discrete error-checking cell are bound probe molecules of a plurality of species, such that each species of probe molecule is bound to one discrete observational cell and to at least one discrete error-checking cell.    
     
     
         52 . The detection array of  claim 51  wherein said hybridization region comprises an oligonucleotide of a length between 4 and 8 nucleotides.  
     
     
         53 . The detection array of  claim 51  wherein said hybridization region comprises a peptido-nucleic acid subsequence.  
     
     
         54 . The detection array of  claim 51  wherein said core region comprises an oligonucleotide having a length and a sequence such that the Tm of said core region hybridized to a complementary oligonucleotide is at least 50° C.  
     
     
         55 . The detection array of  claim 51  wherein said hybridization region comprises an oligonucleotide, and wherein each said error-checking cell has bound a plurality of species of probe molecules, said plurality being collectively degenerate at one or more nucleotide positions in said hybridization region.  
     
     
         56 . The detection array of  claim 51  wherein said discrete observational cells are arranged into one or more substantially rectangular arrays, and wherein said discrete error-checking cells are arranged into substantially linear arrays parallel to said rectangular arrays, said arrangement being such that said each error-checking cell has bound the species of probes which are bound to the perpendicularly adjacent row or column of said rectangular array.  
     
     
         57 . The detection array of  claim 51  wherein said surfaces are planar glass surfaces or controlled pore glass beads derivitized with amino-reactive moieties selected from the group of aldehyde, ketone, thiocyanate, isothiocyanate, and carboxylic acid, and said attachment means comprises an amino functional group for binding with said amino-reactive moieties.  
     
     
         58 . The detection array of  claim 51  wherein said surfaces are planar glass surfaces or controlled pore glass beads derivitized with moieties selected from the group of avidin and streptavidin, and said attachment means comprises a biotin functional group.  
     
     
         59 . The detection array of  claim 51  wherein said surfaces are planar glass surfaces or controlled pore glass beads derivitized with antibodies directed to a hapten, and said attachment means comprises said hapten.  
     
     
         60 . The detection array of  claim 59  wherein said hapten is selected from the group consisting of dinitrophenol and digoxigenin.  
     
     
         61 . The detection array of  claim 51  wherein said surfaces are surfaces of beads and said attachment means comprises a functional group selected from the group consisting of biotin and digoxigenin.  
     
     
         62 . The detection array of  claim 51  wherein attachment means comprise an aliphatic chain.  
     
     
         63 . The detection array of  claim 62  wherein said aliphatic chain has a length of at least 6.  
     
     
         64 . The detection array of  claim 51  further comprising stacking oligomers hybridized with a portion of said sore region, said portion being adjacent to said hybridization regions of said probe molecules.  
     
     
         65 . The detection array of  claim 64  wherein said terminal subsequence of said target nucleic acids comprise first single-stranded end subsequences, and said detection array further comprises said target nucleic acids hybridized with probes having hybridization regions complementary to said first single-stranded subsequences, such that said hybridization regions of said probe molecule are hybridized with said first single-stranded end subsequences of said target nucleic acids.  
     
     
         66 . The detection array of  claim 64  wherein said terminal subsequence of said target nucleic acids comprise first single-stranded end subsequences and adjacent double-stranded subsequences, and said detection array further comprises said target nucleic acids hybridized with probes having a hybridization region complementary to said first single-stranded subsequences and to said adjacent double-stranded subsequences, such that said hybridization region of said probe molecule is hybridized with said first single-stranded end subsequence and with said adjacent double-stranded subsequence of said target nucleic acid.  
     
     
         67 . A method for detecting a terminal subsequence in a target nucleic acid, comprising: 
 (a) hybridizing said target nucleic acid and a stacking oligonucleotide to probe molecules of a universal array of  claim 51 , wherein said target nucleic acid hybridizes to a hybridization region of said probe molecules, wherein said stacking oligonucleotide hybridizes to at least a portion of a core region of said probes, said portion being adjacent to said hybridization region of said probe molecules, and wherein said hybridizing occurs in the presence of a nucleic acid ligase under ligating conditions;    (b) washing the hybridized detection array in denaturing conditions; and    (c) detecting which probe molecules have hybridized with said target nucleic acid.    
     
     
         68 . The method of  claim 67  wherein said terminal subsequence of said target nucleic acid is single-stranded, and wherein said hybridization region of said probe molecules hybridizes to said single-stranded end subsequence.  
     
     
         69 . The method of  claim 68  wherein said denaturing conditions comprise conditions sufficient to denature all double stranded nucleic acids.  
     
     
         70 . The method of  claim 68  wherein said denaturing conditions comprise washing at a temperature greater than 80° C.  
     
     
         71 . The method of  claim 68  wherein said ligating conditions comprise a ligase enzyme concentration at which the amount of hybridized target nucleic acids reaches a plateau.  
     
     
         72 . The method of  claim 68  wherein that strand of said target nucleic acid that is ligated to said probe molecule is labeled with a detectable label.  
     
     
         73 . The method of  claim 67  wherein said terminal subsequence of said target nucleic acid comprises a single-stranded end subsequence and an adjacent double-stranded subsequence, and wherein said hybridization region of said probe molecules hybridizes to said single-stranded end subsequence and to a strand of said adjacent double-stranded subsequence, whereby a strand of said target nucleic acid is displaced from said double-stranded region.  
     
     
         74 . The method of  claim 73  wherein said denaturing conditions comprise conditions incapable of denaturing said stacking oligomer from said probe molecule.  
     
     
         75 . The method of  claim 73  wherein said denaturing conditions comprise washing at a temperature between 40° C. and 65° C.  
     
     
         76 . The method of  claim 73  wherein said ligating conditions comprise conditions such that substantially all nicks in correctly hybridized nucleic acids are ligated.  
     
     
         77 . The method of  claim 73  wherein said stacking oligomer is labeled with a detectable label.  
     
     
         78 . A method for binding probe molecules on a glass surface comprising: 
 (a) preparation of said glass surface comprising washing with an acid of a pH of no more than 1;    (b) amino-reactive-derivitizing said prepared surface with amino-reactive groups;    (c) contacting said derivitized surface with a solution of probe molecules in order to deposit said probe molecules, wherein said solution has a concentration of probe molecules of less than 200 micro-moles per liter, and wherein said probe molecules comprise an amino functional group and a subsequence of at least 16 oligonucleotides; and    (d) passivating amino-reactive groups on said contacted surface.    
     
     
         79 . The method of  claim 78  wherein said acid comprises nitric acid of a concentration of at least 65%.  
     
     
         80 . The method of  claim 78  wherein said amino-reactive groups comprise phenylisothiocyanate groups.  
     
     
         81 . The method of  claim 78  wherein said step of amino-reactive-derivitization comprises: 
 (a) amino-derivitizing said prepared surface with amino groups by immersion in an amino containing silane; and  
 (b) conjugating amino-reactive groups to said amino groups on said surface by immersion in phenylene diisothiocyanate.  
 
     
     
         82 . The method of  claim 81  wherein said amino containing silane has the formula (R 1 R 2 )NR 3 Si(R 4 R 5 R 6 ), wherein R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  comprise aliphatic chains of length from C 1  to C 10 , except that at least one of R 1  and R 2  consists of an H, and except that at least one of R 4 , R 5 , and R 6  consists of the structure R—O—, wherein R is a methyl or an ethyl group.  
     
     
         83 . The method of  claim 82  wherein said amino containing silane is 3′-aminopropyltrimethoxysilane.  
     
     
         84 . The method of  claim 81  wherein immersion conditions of said amino-derivitization step are selected in order that substantially all groups on said glass surfaces reactive with said amino containing silane are reacted with said amino-silane.  
     
     
         85 . The method of  claim 81  wherein immersion conditions of said conjugating step are selected in order that substantially all said amino groups on said glass surface are conjugated with phenylisothiocyanate groups.  
     
     
         86 . The method of  claim 78  wherein said step of contacting comprises contacting with aliquots of probe solution.  
     
     
         87 . The method of  claim 78  wherein said solution of probe molecules comprises a buffer having a pH between 8 and 10.  
     
     
         88 . The method of  claim 78  further comprising, after said step of contacting, a step binding said amino functional groups of said deposited probe molecules with said amino-reactive groups on said glass surface.  
     
     
         89 . The method of  claim 88  wherein said step of binding comprises hydrating by exposing said surface to a humid atmosphere.  
     
     
         90 . A detection array according to  claim 51  wherein probe molecules are bound to a surface according to the method of  claim 78 .  
     
     
         91 . A method for differential gene expression analysis comprising: 
 (a) applying the method of  claim 1  to a nucleic acid sample derived from a first tissue;    (b) applying the method of  claim 1  to a nucleic acid sample derived from a second tissue; and    (c) comparing the nucleic acids identified in step (a) with the nucleic acids identified in step (b).    
     
     
         92 . The method of  claim 91  wherein said first tissue comprises a particular tissue in a first state, and wherein said second tissue comprises said particular tissue in a second state.  
     
     
         93 . A kit comprising in separate containers: 
 (a) first reagents for providing a subsample of first nucleic acid fragments from an original sample of nucleic acids, said first nucleic acid fragments in said subsample being derived from those nucleic acids in said original sample having selected sequences for a first and a second primary nucleotide subsequence;    (b) second reagents for providing second nucleic acid fragments from said subsample of first nucleic acid fragments, wherein said second nucleic acid fragments have an additional subsequence comprising a terminal single-stranded subsequence of said second nucleic acid fragments, and wherein said additional subsequence is at a fixed distance from said first primary subsequence; and    (c) a detection array according to  claim 51  for recognizing said additional subsequences of said second nucleic acid fragments.    
     
     
         94 . The kit of  claim 93  further comprising a computer readable medium containing instructions for causing a computer to search a database of nucleic acid sequences for those sequences having said first primary nucleotide subsequence, second primary nucleotide subsequence, and said additional nucleotide subsequence.  
     
     
         95 . The kit of  claim 93  wherein said first reagents comprise: 
 (a) one or more restriction endonucleases of a type that cut a nucleic acid within their recognition sequences;  
 (b) one or more partially double stranded adapter nucleic acids, each said adapter nucleic acid having a terminal subsequence for hybridizing with the end of a nucleic acid cut by one of said one or more restriction endonucleases; and  
 (c) a ligase enzyme.  
 
     
     
         96 . The kit of  claim 93  wherein said second reagents comprise a restriction endonuclease of a type that cuts a nucleic acid outside of its recognition sequence.  
     
     
         97 . A computer-based system for processing gene-expression information comprising: 
 (a) input/output means for input of user requests and output of processing responses;    (b) storage means for storing nucleic acid sequences identified in samples of nucleic acids according to the method of  claim 1;  and    (c) processing means for, according to said user requests, either 
 (i) searching a database of nucleic acid sequences in order to locate database sequences having said observed subsequence sets or to determine that no such database sequences exist, said database of nucleic acid sequences comprising nucleic acid sequences that might be present in said sample, and storing said located database sequences in said storage means, or  
 (ii) for comparing two or more sequences retrieved from said storage means, said sequences having been identified in two or more samples of nucleic acids, in order to determine differential presence of said identified database sequences in said samples, and generating processing responses of said searching or of said comparing.  
   
     
     
         98 . The computer-based system of  claim 97  wherein said samples comprise nucleic acids derived from two or more tissue samples, and wherein said processing means for comparing determines differential gene expression between said tissue samples.  
     
     
         99 . The computer-based system of  claim 97  wherein said processing means, according to said user requests, further is for searching sequences retrieved from said storage means for those with homology to a user query sequence.