Arrays of Nucleic Acid Probes for Analyzing Biotransformation Genes

Abstract

The invention provides arrays of immobilized probes, and methods employing the arrays, for detecting mutations in the biotransformation genes, such as cytochromes P450. For example, one such array comprises four probe sets. A first probe set comprises a plurality of probes, each probe comprising a segment of at least three nucleotides exactly complementary to a subsequence of a reference sequence from a biotransformation gene, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence. Second, third and fourth probe sets each comprise a corresponding probe for each probe in the first probe set. The probes in the second, third and fourth probe sets are identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least three nucleotides thereof that includes the at least one interrogation position, except that the at least one interrogation position is occupied by a different nucleotide in each of the four corresponding probes from the four probe sets.

What is claimed is: 
     
         1 . An array of nucleic acid probes immobilized on a solid support, the array comprising at least two sets of probes,
 (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of a reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence,   (2) a second probe set comprising a corresponding probe for each probe in the first probe set, the corresponding probe in the second probe set being identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the at least one interrogation position, except that the at least one interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets;   wherein the probes in the first probe set have at least three interrogation positions respectively corresponding to each of three contiguous nucleotides in the reference sequence;   provided that the array does not consist of a complete set of probes of a given length, wherein a complete set is all permutations of nucleotides A, C, G and T/U;   wherein the reference sequence is from a biotransformation gene.   
     
     
         2 . A method of using an addressable array of biopolymers on a substrate, comprising: (a) receiving an array of addressable biopolymer regions and an associated machine readable identifier carried on an array substrate or array housing; (b) exposing the array to a sample; (c) reading the array; (d) machine reading the identifier as an identifier signal; and (e) retrieving updated biological function data for one or more of the biopolymers from a memory based on the identifier signal, wherein the retrieved biological function data comprises information on the function of a target of the array, or its complement, or the gene from which either originated; wherein the retrieval of the biological function data includes: communicating the identifier signal to a processor which retrieves data on the identity of the biopolymers based on the read identifier, and communicating the identity data on the biopolymers to a processor which retrieves the biological function data for one or more of the biopolymers from a memory based on the retrieved identity data. 
     
     
         3 . A method according to  claim 2  wherein the biopolymers are polynucleotides. 
     
     
         4 . A method according to  claim 3  wherein the biopolymers are DNA. 
     
     
         5 . A method according to  claim 2  wherein the memory from which biological function data is retrieved is a portable storage medium received from a remote location. 
     
     
         6 . A method according to  claim 5  wherein the machine readable identifier is read while the array is in a same apparatus which reads the array. 
     
     
         7 . A method according to  claim 2  wherein the processor which retrieves the biological function data and the memory from which the biological function data is retrieved, are remote from the location at which the array and identifier are read, and wherein the read identifier or identity data is communicated to the remote processor. 
     
     
         8 . A method according to  claim 2  wherein the retrieved biological function data comprises information on the gene from which a target or its complement originated. 
     
     
         9 . A method according to  claim 8  wherein the biopolymers are polynucleotides. 
     
     
         10 . A method according to  claim 2  wherein the retrieved biological function data comprises information on the gene from which a target of the array, or its complement, originated. 
     
     
         11 . A method of using an addressable array of biopolymers on a substrate, comprising: (a) receiving an array of addressable biopolymer regions and an associated machine readable identifier carried on an array substrate or array housing; (b) exposing the array to a sample; (c) reading the array; (d) machine reading the identifier as an identifier signal; and (c) communicating with a remote station and retrieving therefrom updated biological function data for one or more of the biopolymers based on the identifier signal, wherein the retrieved biological function data comprises information on the function of a target of the array, or its complement, or the gene from which either originated, wherein the retrieval of the biological function data includes: communicating the identifier signal to a processor which retrieves data on the identity of the biopolymers based on the read identifier; and communicating the identity data on the biopolymers to a processor which retrieves the biological function data for one or more of the biopolymers from a memory based on the retrieved identity data. 
     
     
         12 . A method according to  claim 11  wherein the biological function data is retrieved by communicating to the remote station the identifier signal, or communicating to the remote station, biopolymer identity obtained using the identifier signal, and receiving the biological function data in response. 
     
     
         13 . A method according to  claim 12  additionally comprising: obtaining a communication address of the remote station using the identifier signal; wherein the communication address is used to establish communication with the remote station. 
     
     
         14 . A method according to  claim 12  additionally comprising retrieving the biopolymer identity data from a memory carrying multiple identifiers in association with the biopolymer identity data, using the identifier signal, and wherein the biopolymer identity data is communicated to the remote station to retrieve the biological function data in response. 
     
     
         15 . A method according to  claim 11  wherein the biopolymers are polynucleotides. 
     
     
         16 . An array of oligonucleotide probes immobilized on a solid support, the array comprising at least one pair of first and second probe groups, each group comprising a first and second sets of oligonucleotide probes as defined by  claim 16 ; wherein each probe in the first probe set from the first group is exactly complementary to a subsequence of a first reference sequence and each probe in the first probe set from the second group is exactly complementary to a subsequence from a second reference sequence. 
     
     
         17 . The array of  claim 16 , wherein each group further comprises third and fourth probe sets, each comprising a corresponding probe for each probe in the first probe set, the probes in the second, third and fourth probe sets being identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least three nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the four corresponding probes from the four probe sets. 
     
     
         18 . The array of  claim 16 , wherein the first reference sequence includes the site of a mutation in the biotransformation gene, and the second reference sequence includes a site of a silent polymorphism within the biotransformation gene or flanking the biotransformation gene. 
     
     
         19 . The array of  claim 18 , wherein the reference sequence is from a gene encoding an enzyme selected from the group consisting of a cytochrome P450, N-acetyl transferase II, glucose 6-phosphate dehydrogenase, pseudocholinesterase, catechol-O-methyl transferase, and dihydropyridine dehydrogenase. 
     
     
         20 . The array of  claim 18  that comprises at least forty pairs of first and second probe groups, wherein the probes in the first probe sets from the first groups of the forty pairs are exactly complementary to subsequences from forty respective first reference sequences. 
     
     
         21 . A block of oligonucleotide probes immobilized on a solid support, comprising: a perfectly matched probe comprising a segment of at least three nucleotides exactly complementary to a subsequence of a reference sequence, the segment having a plurality of interrogation positions respectively corresponding to a plurality of nucleotides in the reference sequence, for each interrogation position, three mismatched probes, each identical to a sequence comprising the perfectly matched probe or a subsequence of at least three nucleotides thereof including the plurality of interrogation positions, except in the interrogation position, which is occupied by a different nucleotide in each of the three mismatched probes and the perfectly matched probe; provided the array lacks a complete set of probes of a given length; wherein the reference sequence is from a biotransformation gene. 
     
     
         22 . The array of  claim 20 , wherein the segment of the perfectly matched probe comprises 3-20 interrogation positions corresponding to 3-20 respective nucleotides in the reference sequence. 
     
     
         23 . An array of probes immobilized to a solid support comprising at least two blocks of probes, each block as defined by  claim 20 , a first block comprising a perfectly matched probe comprising a segment exactly complementary to a subsequence of a first reference sequence and a second block comprising a perfectly matched probe comprising a segment exactly complementary to a subsequence of a second reference sequence. 
     
     
         24 . The array of  claim 23 , wherein the first reference sequence is from a wildtype 2D6 gene and the second reference sequence is from a mutant 2D6 gene. 
     
     
         25 . The array of  claim 23 , comprising at least 10-100 blocks of probes, each comprising a perfectly matched probe comprising a segment exactly complementary to a subsequence of at least 10-100 respective reference sequences. 
     
     
         26 . An array of oligonucleotide probes immobilized on a solid support, the array comprising at least four probes: a first probe comprising first and second segments, each of at least three nucleotides and exactly complementary to first and second subsequences of a reference sequence, the segments including at least one interrogation position corresponding to a nucleotide in the reference sequence, wherein either (1) the first and second subsequences are noncontiguous, or (2) the first and second subsequences are contiguous and the first and second segments are inverted relative to the complement of the first and second subsequences in the reference sequence; second, third and fourth probes, identical to a sequence comprising the first probe or a subsequence thereof comprising at least three nucleotides from each of the first and second segments, except in the at least one interrogation position, which differs in each of the probes; provided the array lacks a complete set of probes of a given length; wherein the reference sequence is from a biotransformation gene. 
     
     
         27 . A method of comparing a target nucleic acid with a reference sequence comprising a predetermined sequence of nucleotides, the method comprising: (a) hybridizing the target nucleic acid to an array of oligonucleotide probes immobilized on a solid support, the array comprising: a perfectly matched probe comprising a segment of at least three nucleotides exactly complementary to a subsequence of a reference sequence, the segment having a plurality of interrogation positions respectively corresponding to a plurality of nucleotides in the reference sequence, wherein the reference sequence is from a biotransformation gene; for each interrogation position, three mismatched probes, each identical to a sequence comprising the perfectly matched probe or a subsequence of at least three nucleotides thereof including the plurality of interrogation positions, except in the interrogation position, which is occupied by a different nucleotide in each of the three mismatched probes and the perfectly matched probe; (b) for each interrogation position, (1) comparing the relative specific binding of the three mismatched probes and the perfectly matched probe; (2) assigning a nucleotide in the target sequence as the complement of the interrogation position of the probe having the greatest specific binding. 
     
     
         28 . The method of  claim 27 , wherein the target sequence has an undetermined substitution relative to the reference sequence, and the method assigns a nucleotide to the substitution. 
     
     
         29 . A method of screening a patient for capacity to metabolize a drug, the method comprising: (a) hybridizing a tissue sample from the patient containing a target nucleic acid to an array of oligonucleotide probes immobilized on a solid support, the array comprising: (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least three nucleotides exactly complementary to a subsequence of the reference sequence from a biotransformation gene which metabolizes the drug, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence, (2) a second probe set comprising a corresponding probe for each probe in the first probe set, the corresponding probe in the second probe set being identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least three nucleotides thereof that includes the at least one interrogation position, except that the at least one interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets; wherein, the probes in the first probe set have at least three interrogation positions respectively corresponding to each of at least three nucleotides in the reference sequence, and (b) determining which probes, relative to one another, in the first and second probe sets specifically to the target nucleic acid, the relative specific binding of corresponding probes in the first and second probe sets indicating whether the target sequence contains a mutation relative to the reference sequence, which, if present, impairs the capacity of the patient to metabolize the drug. 
     
     
         30 . A method of conducting a clinical trial on a drug, the method comprising: (a) obtaining a tissue sample containing a target nucleic acid from each of a pool of patients; (b) for each tissue sample, hybridizing the target nucleic acid to an array of oligonucleotide probes immobilized on a solid support, the array comprising: (1) a first probe set comprising a plurality of probes, each probe comprising a segment of at least three nucleotides exactly complementary to a subsequence of the reference sequence from a biotransformation gene, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence, (2) a second probe set comprising a corresponding probe for each probe in the first probe set, the corresponding probe in the second probe set being identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least three nucleotides thereof that includes the at least one interrogation position, except that the at least one interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets; wherein, the probes in the first probe set have at least three interrogation positions respectively corresponding to each of at least three nucleotides in the reference sequence; (c) determining which probes, relative to one another, in the first and second probe sets specifically to the target nucleic acid, the relative specific binding of corresponding probes in the first and second probe sets indicating whether the target sequence contains a mutation relative to the reference sequence selecting a subpool of patients having a target sequence free of the mutation; and (d) administering the drug to the subpool of patients to determine efficacy. 
     
     
         31 . The method of  claim 30 , further comprising combining the drug with a pharmaceutical carrier to form a pharmaceutical composition.