Arrays of nucleic acid probes for detecting cystic fibrosis
The invention provides arrays of immobilized probes, and methods employing the arrays, for detecting mutations in the CFTR gene.
1 - 30 . (canceled)
31 . A device for detecting at least one variation in the splicing of a gene comprising
an array of nucleic acid probes immobilized on a solid support, the array comprising at least two sets of probes of between 3 and 100 nucleotides in length,
wherein said array comprises at least a first and a second probe,
wherein said first probe comprises a first sequence that is complementary to an exon or an intron of a gene, and wherein said sequence corresponds to at least one region of variation corresponding to a splice sequence, and
wherein said second probe comprises a second sequence that is complementary to an exon-intron boundary of said gene, and wherein said second sequence corresponds to at least one region of variation corresponding to a splice sequence,
said device allowing, when hybridized with a target sequence, detection of the presence or absence of said at least one variation in the splicing of a gene.
32 . The device of claim 31 , wherein said probe sequences are publicly available.
33 . The device of claim 31 , wherein the probes are immobilized on a chip.
34 . The device of claim 31 , wherein said probes are oligodeoxyribonucleotides or oligoribonucleotides.
35 . The device of claim 31 , wherein said probes comprise sequences of between 3 and 50 nucleotides.
36 . The device of claim 31 , wherein said first and second probes exhibit complementarity to reference sequences comprising mutations or polymorphisms associated with phenotypic changes having clinical significance in human patients.
37 . The device of claim 36 , wherein said first and second probes exhibit complementarity to reference sequences comprising mutations or polymorphisms associated with cancer.
38 . A method of producing a device comprising an array of nucleic acid probes immobilized on a solid support, the array comprising at least two sets of probes of between 3 and 100 nucleotides in length,
(a) providing said nucleic acid probes, wherein said probes comprise at least a first and a second probe,
wherein said first probe comprises a first sequence that is complementary to an exon or an intron of a gene, and wherein said sequence corresponds to at least one region of variation corresponding to a splice sequence, and
wherein said second probe comprises a second sequence that is complementary to an exon-intron boundary of said gene, and wherein said second sequence corresponds to at least one region of variation corresponding to a splice sequence; and
(b) arranging and immobilizing said first and second probes adjacent to one another on the solid support,
said device allowing, when hybridized with a target sequence, detection of the presence or absence of said at least one variation in the splicing of a gene.
39 . The method of claim 38 , wherein said first or second probe is obtained by:
(a) identifying at least two nucleic acid sequences corresponding to a splice sequence and a mutation in a splice sequence, respectively, wherein said mutation has a phenotypic effect of clinical significance, and
(b) synthesizing nucleic acid probes containing complementarity to said splice sequence.
40 . The method of claim 38 , wherein said probe sequences are publicly available.
41 . The method of claim 38 , wherein the probes are immobilized on a chip.
42 . The method of claim 38 , wherein said first and second probes exhibit complementarity to reference sequences comprising mutations or polymorphisms associated with phenotypic changes having clinical significance in human patients.
43 . The method of claim 42 , wherein said first and second probes exhibit complementarity to reference sequences comprising mutations or polymorphisms associated with cancer.
44 . The method of claim 38 , wherein said probes comprise sequences of between 3 and 50 nucleotides.
45 . The device of claim 31 , wherein said device allows detection of the presence or absence of said at least one variation in the splicing of a gene in an mRNA population.
46 . The device of claim 31 , wherein said device allows detection of the presence or absence of at least one variation in the splicing of more than one gene.
47 . A device for identifying at least one differentially spliced gene product, wherein said device comprises:
a solid support material and single-stranded oligonucleotides of between 5 and 100 nucleotides in length attached to said support material,
wherein said oligonucleotides comprise at least a first and a second oligonucleotide molecule arranged serially on the support material,
wherein said first oligonucleotide molecule comprises a first sequence that is complementary to and specific for an exon or an intron of a first gene, and wherein said first sequence corresponds to a region of variability in at least one product of said first gene due to differential splicing, and
wherein said second oligonucleotide molecule comprises a second sequence that is complementary to and specific for an exon-exon or exon-intron junction region of said first gene, and wherein said second sequence corresponds to a region of variability in at least one product of said first gene due to differential splicing,
said device allowing, when contacted with a sample containing at least one nucleic acid molecule under conditions allowing hybridisation to occur, the determination of the presence or absence of said differentially spliced gene product.
48 . The device of claim 47 , wherein said first and second oligonucleotide molecules are available from a compilation of published sequences or sequence information from at least one database.
49 . The device of claim 47 , wherein the support material is selected from the group consisting of a filter, a membrane and a chip.
50 . The device of claim 47 , wherein said single-stranded oligonucleotides are RNA or DNA molecules.
51 . The device of claim 47 , wherein said single-stranded oligonucleotides comprise oligonucleotides of less than 50 nucleotides in length.
52 . The device of claim 47 , wherein said single-stranded oligonucleotides are specific for alternative splicings representative of a cell or tissue in a given pathological condition.
53 . The device of claim 52 , wherein said single-stranded oligonucleotides are specific for alternative splicings representative of a tumor cell or tissue.
54 . The device of claim 52 , wherein said single-stranded oligonucleotides are specific for alternative splicings representative of a cell or tissue undergoing apoptosis.
55 . The device of claim 47 , where said device is useful to evaluate the toxicity of a compound or treatment to a cell, tissue, or organism by determining the presence or absence of said differentially spliced gene product in a sample treated with said compound or treatment.
56 . The device of claim 47 , where said device is useful to evaluate the therapeutic efficacy of a compound to a cell, tissue, or organism by determining the presence or absence of said differentially spliced gene product in a sample from said cell, tissue, or organism.
57 . The device of claim 47 , where said device is useful to evaluate the responsiveness of a subject to a compound or treatment by determining the presence or absence of said differentially spliced gene product in a sample from said subject exposed to said compound or treatment.
58 . A method of producing a device comprising a support material and single-stranded oligonucleotide of between 5 and 100 nucleotides in length attached to said solid support material, wherein said method comprises:
(a) providing said oligonucleotides, wherein said oligonucleotides comprise at least a first and a second oligonucleotide molecule,
wherein said first oligonucleotide molecule comprises a first sequence that is complementary to and specific for an exon or an intron of a first gene, and wherein said first sequence corresponds to a region of variability in at least one product of said first gene due to differential splicing, and
wherein said second oligonucleotide molecule comprises a second sequence that is complementary to and specific for an exon-exon or exon-intron junction region of said first gene, and wherein said second sequence corresponds to a region of variability in at least one product of said first gene due to differential splicing; and
(b) arranging and immobilizing said oligonucleotides serially on said support material,
said device allowing, when contacted with a sample containing at least one nucleic acid molecule under conditions allowing hybridisation to occur, the determination of the presence or absence of at least one differentially spliced gene product.
59 . The method of claim 58 , wherein said first or second oligonucleotide molecule is obtained by a method comprising:
(a) identifying at least two different oligonucleotides corresponding to a differentially spliced domain of a gene, wherein said differentially spliced domain is characteristic of a physiopathological condition, and
(b) synthesizing one or several single-stranded oligonucleotides complementary to and specific for said domain or a junction region formed by the splicing or absence of splicing of said domain.
60 . The method of claim 59 , wherein the identification step (a) comprises:
i) hybridizing a plurality of different RNA or cDNA molecules derived from a first sample, wherein the composition or sequence of the RNA or cDNA molecules is at least partially unknown, with a plurality of different cDNA molecules derived from RNA molecules of a second sample, wherein the composition or sequence of the cDNA molecules is at least partially unknown; and
ii) identifying, from the hybrids formed in i), a population of nucleic acid molecules comprising an unpaired region, wherein said unpaired region corresponds to a region of a gene that is differentially spliced between said first and second sample.
61 . The method of claim 58 , wherein said first and second oligonucleotide molecules are obtained from a compilation of published sequences or sequence information from databases.
62 . The method of claim 58 , wherein the support material is selected from a filter, a membrane, and a chip.
63 . The method of claim 58 , wherein said single-stranded oligonucleotides are specific for alternative splicings representative of a cell or tissue in a given pathological condition.
64 . The method of claim 63 , wherein said single-stranded oligonucleotides are specific for alternative splicings representative of a tumor cell or tissue.
65 . The method of claim 63 , wherein said single-stranded oligonucleotides are specific for alternative splicings representative of a cell or tissue undergoing apoptosis.
66 . The method of claim 58 , wherein said single-stranded oligonucleotides comprise oligonucleotides of less than 50 nucleotides in length.
67 . The device of claim 47 , wherein said device allows the determination of the presence or absence of two or more differentially spliced gene products of said first gene.
68 . The device of claim 47 , wherein said device allows the determination of the presence or absence of one or more differentially spliced gene products of two or more genes.