Nucleic acid sequencing processes using non-radioactive detectable modified or labeled nucleotides or nucleotide analogs, and other processes for nucleic acid detection and chromosomal characterization using such non-radioactive detectable modified or labeled nucleotides or nucleotide analogs
DC CAFCFirst Claim
1. A process for detecting a nucleic acid of interest in a sample, which process comprises:
- (A) providing a sample which may contain a nucleic acid of interest;
(B) providing;
(i) an oligo- or polynucleotide that comprises two segments, the first segment comprising a nucleotide sequence that is complementary to and capable of specifically hybridizing to and forming a hybrid with said nucleic acid of interest or a portion thereof, and the second segment comprising an operator sequence that is capable of binding to or complexing with a non-radioactively detectable protein; and
(ii) a non-radioactively detectable protein which is non-radioactive and has a binding affinity to said operator sequence;
(C) contacting a sample suspected of containing said nucleic acid of interest with said oligo- or polynucleotide (i) and said non-radioactively detectable protein (ii) to form a complex; and
(D) detecting non-radioactively the presence of said non-radioactively detectable protein in said complex to detect said nucleic acid of interest.
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Abstract
A process for determining the sequence of nucleic acids of interest employs nucleotides or nucleotide analogs that have been made detectable by non-radioactive modifying or labeling. Such nucleotides or nucleotide analogs are modified on the sugar moieties, the phosphate moieties or the base moieties, including base analogs. Modified nucleotide analogs can be attached to or coupled to or incorporated into DNA or RNA. The modified or labeled nucleotides or nucleotide analogs are also useful in processes for detecting the presence of nucleic acids of interest and for characterizing chromosomal sequences. Detection processes using the modified or labeled nucleotides or nucleotide analogs extend to the use of a gel for separating or resolving hybrids formed between non-radioactively labeled oligonucleotides or polynucleotides and such nucleic acids of interest.
75 Citations
198 Claims
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1. A process for detecting a nucleic acid of interest in a sample, which process comprises:
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(A) providing a sample which may contain a nucleic acid of interest; (B) providing; (i) an oligo- or polynucleotide that comprises two segments, the first segment comprising a nucleotide sequence that is complementary to and capable of specifically hybridizing to and forming a hybrid with said nucleic acid of interest or a portion thereof, and the second segment comprising an operator sequence that is capable of binding to or complexing with a non-radioactively detectable protein; and (ii) a non-radioactively detectable protein which is non-radioactive and has a binding affinity to said operator sequence; (C) contacting a sample suspected of containing said nucleic acid of interest with said oligo- or polynucleotide (i) and said non-radioactively detectable protein (ii) to form a complex; and (D) detecting non-radioactively the presence of said non-radioactively detectable protein in said complex to detect said nucleic acid of interest. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62)
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63. A process for determining whether the number of copies of a particular chromosome in a cell is normal or abnormal, the process comprising:
- providing at least one cell;
contacting said cell under hybridizing conditions with one or more clones or DNA fragments, or oligo- or polynucleotides derived from said clone or clones, wherein said clones or fragments or oligo- or polynucleotides are capable of hybridizing specifically to a locus or loci of said particular chromosome or a portion thereof, wherein said clones or fragments or oligo- or polynucleotides comprise one or more detectable non-radioactive modified or labeled nucleotides or one or more detectable non-radioactively modified or labeled nucleotide analogs, which nucleotide analogs can be attached to or coupled to or incorporated into DNA or RNA, and wherein said modified or labeled nucleotides or modified or labeled nucleotide analogs comprise;(i) a nucleotide structure or nucleotide analog structure having the formula
PM-SM-BASE-Sigwherein PM is a phosphate moiety, SM is a furanosyl moiety, BASE is a pyrimidine, a purine, or a 7-deazapurine base moiety; and Sig is a detectable non-radioactive moiety, wherein PM is covalently attached to SM, BASE is covalently attached to SM, and Sig is covalently attached to BASE at a position other than the C5 position when BASE is a pyrimidine moiety, at a position other than the C8 position when BASE is a purine moiety, and at a position other than the C7 position when BASE is a 7-deazapurine moiety; (ii) a nucleotide structure or nucleotide analog structure having the formula - View Dependent Claims (73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 181)
- providing at least one cell;
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64. A process for identifying a chromosome of interest in a cell containing other chromosomes, the process comprising:
- providing at least one cell;
providing a set of clones or DNA fragments, or oligo- or polynucleotides derived from said clone or clones, wherein said clones or fragments or oligo- or polynucleotides are specifically hybridizable to a locus or loci in said chromosome of interest, wherein said clones or fragments or said oligo- or polynucleotides comprise one or more detectable non-radioactive modified or labeled nucleotides or one or more detectable non-radioactively modified or labeled nucleotide analogs, which nucleotide analogs can be attached to or coupled to or incorporated into DNA or RNA, and wherein said modified or labeled nucleotides or modified or labeled nucleotide analogs comprise;(i) a nucleotide structure or nucleotide analog structure having the formula
PM-SM-BASE-Sigwherein PM is a phosphate moiety, SM is a furanosyl moiety, BASE is a pyrimidine, a purine, or a 7-deazapurine base moiety, and Sig is a detectable non-radioactive moiety, wherein PM is covalently attached to SM, BASE is covalently attached to SM, and Sig is covalently attached to BASE at a position other than the C5 position when BASE is a pyrimidine moiety, at a position other than the C8 position when BASE is a purine moiety, and at a position other than the C7 position when BASE is a 7-deazapurine moiety; (ii) a nucleotide structure or nucleotide analog structure having the formula
- providing at least one cell;
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65. A process for identifying a plurality or all of the chromosomes in a cell of interest, the process comprising:
- providing at least one cell;
providing sets of clones or DNA fragments, or oligo- or polynucleotides derived from said clones, wherein said clones or fragments or said oligo- or polynucleotides are capable of hybridizing specifically to a locus or loci in a chromosome of said cell of interest, wherein each of said clones or DNA fragments or oligo- or polynucleotides in said sets are labeled with a different indicator moiety and each of said clones or DNA fragments or oligo- or polynucleotides comprises one or more detectable non-radioactive modified or labeled nucleotides or one or more detectable non-radioactive modified or labeled nucleotide analogs, which nucleotide analogs can be attached to or coupled to or incorporated into DNA or RNA, and wherein said modified or labeled nucleotides or modified or labeled nucleotide analogs comprise;(i) a nucleotide structure or nucleotide analog structure having the formula
PM-SM-BASE-Sigwherein PM is a phosphate moiety, SM is a furanosyl moiety, BASE is a pyrimidine, a purine, or a 7-deazapurine base moiety, and Sig is a detectable non-radioactive moiety, wherein PM is covalently attached to SM, BASE is covalently attached to SM, and Sig is covalently attached to BASE at a position other than the C5 position when BASE is a pyrimidine, at a position other than the C8 position when BASE is a purine, and at a position other than the C7 position when BASE is a 7-deazapurine; (ii) a nucleotide structure or nucleotide analog structure having the formula - View Dependent Claims (66)
- providing at least one cell;
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67. A process for determining the number of chromosomes in an interphase cell of interest, the process comprising:
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providing at least one interphase cell; providing sets of clones or DNA fragments or oligo- or polynucleotides derived from said clones, wherein said set of clones or DNA fragments or oligo- or polynucleotides are specifically complementary to or specifically hybridizable with at least one locus or loci in a chromosome of said interphase cell of interest and each of said clones or DNA fragments or oligo- or polynucleotides in said sets comprises one or more detectable non-radioactive modified or labeled nucleotides or detectable non-radioactively modified or labeled nucleotide analogs, which nucleotide analogs can be attached to or coupled to or incorporated into DNA or RNA, and wherein said modified or labeled nucleotides or modified or labeled nucleotide analogs comprise one or more of; (i) a nucleotide structure or nucleotide analog structure having the formula
PM-SM-BASE-Sigwherein PM is a phosphate moiety, SM is a furanosyl moiety, BASE is a pyrimidine, a purine, or a 7-deazapurine base moiety, and Sig is a detectable non-radioactive moiety, wherein PM is covalently attached to SM, BASE is covalently attached to SM, and Sig is covalently attached to BASE at a position other than the C5 position when BASE is a pyrimidine moiety, at a position other than the C8 position when BASE is a purine, and at a position other than the C7 position when BASE is a 7-deazapurine; (ii) a nucleotide structure or nucleotide analog structure having the formula - View Dependent Claims (68, 69, 70, 71, 72)
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156. A process for detecting a nucleic acid of interest in a sample, which process comprises:
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(a) providing a sample which may comprise a nucleic acid of interest; (b) providing a metal or metal ion; (c) specifically hybridizing said nucleic acid of interest in the sample with one or more oligo- or polynucleotides, each such oligo- or polynucleotide being complementary to or capable of hybridizing with said nucleic acid of interest or a portion thereof, wherein said oligo- or polynucleotides comprise one or more detectable non-radioactive modified or labeled nucleotides or detectable non-radioactive modified or labeled nucleotide analogs, which nucleotide analogs can be attached to or coupled to or incorporated into DNA or RNA, and wherein said modified or labeled nucleotides or modified or labeled nucleotide analogs comprise a nucleotide structure or nucleotide analog structure comprising; (i) a nucleotide structure or nucleotide analog structure having the formula
PM-SM-BASE-Sigwherein PM is a phosphate moiety, SM is a furanosyl moiety, BASE is a pyrimidine, a purine or a 7-deazapurine base moiety; and Sig is a signalling moiety comprising a chelating structure or component capable of chelating said metal or metal ion and providing a detectable signal, wherein Sig comprises at least three carbon atoms, wherein PM is covalently attached to SM, BASE is covalently attached to SM, and Sig is covalently attached to BASE directly or through a linkage group at a position other than the C5 position when BASE is a pyrimidine moiety, at a position other than the C8 position when BASE is a purine moiety and at a position other than the C7 position when BASE is a 7-deazapurine moiety, and such covalent attachment does not substantially interfere with double helix formation or nucleic acid hybridization; (ii) a nucleotide structure or nucleotide analog structure having the formula - View Dependent Claims (157, 174, 175, 176, 177, 178, 179, 180, 182, 183, 184, 185, 186, 187)
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158. A process for detecting a nucleic acid of interest in a sample, which process comprises:
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(A) providing; (i) an oligo- or polynucleotide having two segments; (a) a first segment complementary to and capable of hybridizing to a portion of said nucleic acid of interest; and (b) a second segment comprising at least one protein binding sequence; and (ii) a metal or metal ion; (iii) a detectable protein capable of binding to said protein binding sequence and comprising a chelating structure or chelating component capable of chelating said metal or metal ion and providing a detectable signal; (B) contacting a sample suspected of containing said nucleic acid of interest with said oligo- or polynucleotide and said detectable protein (ii) to form a complex; (C) detecting the presence of said protein in said complex and said nucleic acid of interest by means of said metal or metal ion chelated by said chelating structure or chelating component. - View Dependent Claims (173)
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159. A process for determining whether the number of copies of a particular chromosome in a cell is normal or abnormal, the process comprising:
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providing a cell; providing a metal or metal ion; contacting said cell under hybridizing conditions with one or more clones or DNA fragments, or oligo- or polynucleotides derived from said clone or clones, wherein said clones or fragments or oligo- or polynucleotides are capable of hybridizing specifically to a locus or loci of said particular chromosome or a portion thereof, wherein said clones or fragments or oligo- or polynucleotides comprise one or more detectable non-radioactive modified or labeled nucleotides or detectable non-radioactive modified or labeled nucleotide analogs, which nucleotide analogs can be attached to or coupled to or incorporated into DNA or RNA, and wherein said modified or labeled nucleotides or modified or labeled nucleotide analogs comprise a nucleotide structure or nucleotide analog structure comprising; (i) a nucleotide structure or nucleotide analog structure having the formula
PM-SM-BASE-Sigwherein PM is a phosphate moiety, SM is a furanosyl moiety, BASE is a pyrimidine, a purine, or a 7-deazapurine base moiety, and Sig is a signalling moiety comprising a chelating structure or chelating component capable of chelating said metal or metal ion and providing a detectable signal, wherein PM is covalently attached to the SM, BASE is covalently attached to SM, and Sig is covalently attached to BASE at a position other than the C5 position when BASE is a pyrimidine moiety, at a position other than the C8 position when BASE is a purine moiety, and at a position other than the C7 position when BASE is a 7-deazapurine moiety; (ii) a nucleotide structure or nucleotide analog structure having the formula - View Dependent Claims (160)
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161. A process for identifying a chromosome of interest in a cell containing other chromosomes, the process comprising:
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providing a cell; providing a metal or metal ion; providing a set of clones or DNA fragments, or oligo- or polynucleotides derived from said clone or clones, wherein said clones or fragments or oligo- or polynucleotides are specifically hybridizable to a locus or loci in said chromosome of interest, wherein said clones or fragments or oligo- or polynucleotides comprise one or more detectable modified or labeled nucleotides or detectable modified or labeled nucleotide analogs, which nucleotide analogs can be attached to or coupled to or incorporated into DNA or RNA, and wherein said modified or labeled nucleotides or modified or labeled nucleotide analogs comprise a nucleotide structure or nucleotide analog structure comprising; (i) a nucleotide structure or nucleotide analog structure having the formula
PM-SM-BASE-Sigwherein PM is a phosphate moiety, SM is a furanosyl moiety, BASE is a pyrimidine, a purine, or a 7-deazapurine base moiety, and Sig is a signalling moiety comprising a chelating structure or chelating component capable of chelating said metal or metal ion and providing a detectable signal, wherein Sig comprises at least three carbon atoms, wherein PM is covalently attached to SM, BASE is covalently attached to SM, and Sig is covalently attached to BASE at a position other than the C5 position when BASE is a pyrimidine moiety, at a position other than the C8 position when BASE is a purine moiety, and at a position other than the C7 position when BASE is a 7-deazapurine moiety; (ii) a nucleotide structure or nucleotide analog structure having the formula
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162. A process for identifying a plurality or all of the chromosomes in a cell of interest, the process comprising:
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providing a cell of interest; providing a metal or metal ion; providing sets of clones or DNA fragments, or oligo- or polynucleotides derived from said clones, wherein each of said set of clones or DNA fragments or oligo- or polynucleotides are specifically hybridizable to a locus or loci in a chromosome of said cell of interest, wherein each of said clones or DNA fragments or oligo- or polynucleotides comprise one or more detectable modified or labeled nucleotides or detectable modified or labeled nucleotide analogs capable of detection, which nucleotide analogs can be attached to or coupled to or incorporated into DNA or RNA and wherein each set comprises a different indicator molecule, and wherein said modified or labeled nucleotide or modified or labeled nucleotide analogs comprise a nucleotide structure or nucleotide analog structure comprising; (i) a nucleotide structure or nucleotide analog structure having the formula
PM-SM-BASE-Sigwherein PM is a phosphate moiety, SM is a furanosyl moiety, BASE is a pyrimidine, a purine, or a 7-deazapurine base moiety, and Sig is a signalling moiety comprising a chelating structure or chelating component capable of chelating said metal or metal ion and providing a detectable signal, wherein Sig comprises at least three carbon atoms, wherein PM is covalently attached to SM, BASE is covalently attached to SM, and Sig is covalently attached to BASE at a position other than the C5 position when BASE is a pyrimidine, at a position other than the C8 position when BASE is a purine, and at a position other than the C7 position when BASE is a 7-deazapurine; (ii) a nucleotide structure or nucleotide analog structure having the formula
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163. A process for determining the number of chromosomes in an interphase cell of interest, the process comprising:
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providing an interphase cell of interest; providing a metal or metal ion; providing sets of clones or DNA fragments, or oligo- or polynucleotides derived from said clones, wherein each of said set of clones or DNA fragments or oligo- or polynucleotides are specifically complementary to or specifically hybridizable with at least one locus or loci in a chromosome of said interphase cell of interest, wherein each of said clones or DNA fragments or oligo- or polynucleotides in said sets comprise one or more detectable modified or labeled nucleotides or detectable modified or labeled nucleotide analogs, which nucleotide analogs can be attached to or coupled to or incorporated into DNA or RNA, and wherein said modified or labeled nucleotide or modified or labeled nucleotide analog comprise a nucleotide structure or nucleotide analog structure comprising; (i) a nucleotide structure or nucleotide analog structure having the formula
PM-SM-BASE-Sigwherein PM is a phosphate moiety, SM is a furanosyl moiety, BASE is a pyrimidine, a purine, or a 7-deazapurine base moiety, and Sig is a signalling moiety comprising a chelating structure or chelating component capable of chelating said metal or metal ion and providing a detectable signal, wherein PM is covalently attached to SM, BASE is covalently attached to SM, and Sig is covalently attached to BASE at a position other than the C5 position when BASE is a, pyrimidine moiety, at a position other than the C8 position when BASE is a purine, and at a position other than the C7 position when BASE is a 7-deazapurine; (ii) a nucleotide structure or nucleotide analog structure having the formula
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164. A process for preparing a labeled oligo- or polynucleotide of interest, comprising:
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(A) providing a metal or metal ion; (B) providing; (1) one or more detectable chemically modified or labeled nucleotides or detectable chemically modified or labeled nucleotide analogs, which nucleotide analogs can be attached to or coupled to or incorporated into DNA or RNA or an oligo- or polynucleotide of interest, alone or in conjunction with one or more other modified or unmodified nucleic acids selected from the group consisting of nucleotides, oligonucleotides and polynucleotides, wherein said other modified or unmodified nucleic acids are capable of incorporating into an oligo- or polynucleotide of interest, and wherein said modified or labeled nucleotides or nucleotide analogs comprise one or more signalling moieties comprising a chelating structure or chelating component capable of chelating a metal or metal ion and providing a detectable signal, (2) an oligo- or polynucleotide comprising one or more of said modified or labeled nucleotides or modified or labeled nucleotide analogs (1), alone or in conjunction with one or more other modified or unmodified nucleic acids selected from the group consisting of nucleotides, oligonucleotides and polynucleotides, or (3) both (1) and (2), wherein said modified or labeled nucleotides or modified or labeled nucleotide analogs (1) are modified on the furanosyl moiety, the phosphate moiety, the base moiety or any combination thereof, and wherein the modified or labeled nucleotides or modified or labeled nucleotide analogs comprise a nucleotide structure or nucleotide analog structure comprising;
PM-SM-BASE-Sig
(i)wherein PM is a phosphate moiety, SM is a furanosyl moiety, BASE is a pyrimidine, a purine or a 7-deazapurine base moiety, and Sig is a signalling moiety comprising a chelating structure or chelating component capable of chelating said metal or metal ion and providing a detectable signal, wherein Sig comprises at least three carbon atoms, and wherein PM is covalently attached to SM, BASE is covalently attached to SM, and Sig is covalently attached to BASE directly or through a linkage group at a position other than the C5 position when BASE is a pyrimidine moiety, at a position other than the C8 position when BASE is a purine moiety, and at a position other than the C7 position when BASE is a 7-deazapurine moiety; (ii)
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165. A process for detecting the presence of a nucleic acid of interest in a sample, comprising:
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providing a sample which may contain a nucleic acid of interest; providing or generating (i) one or more detectable non-radioactively labeled oligonucleotides or polynucleotides, each of said detectable non-radioactively labeled oligonucleotides or polynucleotides comprising a sequence sufficiently complementary to said nucleic acid of interest or to a portion thereof to specifically hybridize therewith, wherein said detectable non-radioactively labeled oligonucleotides or polynucleotides comprise one or more detectable non-radioactively modified or labeled nucleotides or detectable non-radioactively modified or labeled nucleotide analogs, which nucleotide analogs can be attached to or coupled to or incorporated into DNA or RNA, and (ii) a sample that may contain said nucleic acid of interest; forming in liquid phase hybrids comprising said detectable non-radioactively labeled oligonucleotides or polynucleotides specifically hybridized with said nucleic acid of interest; separating or resolving in a gel said formed hybrids; and detecting non-radioactively the separated or resolved hybrids to detect the presence of said nucleic acid of interest. - View Dependent Claims (166, 167, 168, 169, 170, 171, 172)
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188. A process for detecting the presence of a nucleic acid of interest in a sample, comprising:
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providing or generating (i) one or more detectable non-radioactively labeled oligonucleotide or polynucleotide, each of said detectable non-radioactively labeled oligonucleotide or polynucleotide comprising a sequence sufficiently complementary to said nucleic acid of interest or to a portion thereof to specifically hybridize therewith, wherein said detectable non-radioactively labeled oligonucleotides or polynucleotides comprise one or more detectable non-radioactively modified or labeled nucleotides or detectable non-radioactively modified or labeled nucleotide analogs, which nucleotide analogs can be attached to, coupled to, or incorporated into DNA or RNA, and (ii) a sample that may contain said nucleic acid of interest; forming liquid phase hybrids comprising said detectable non-radioactively labeled oligonucleotides or polynucleotides specifically hybridized with said nucleic acid of interest; subjecting said liquid phase to nuclease treatment to digest non-hybridized single-stranded detectable non-radioactively labeled oligonucleotides or polynucleotides and leave said hybrids intact; and detecting the hybrids non-radioactively to detect the presence of said nucleic acid of interest. - View Dependent Claims (192, 193, 194, 195, 196, 197, 198)
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189. A process for detecting the presence of a nucleic acid of interest in a sample, comprising:
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providing or generating (i) a detectable non-radioactively labeled oligonucleotide or polynucleotide, said detectable non-radioactively labeled oligonucleotide or polynucleotide comprising a sequence sufficiently complementary to said nucleic acid of interest or to a portion thereof to specifically hybridize therewith, wherein said detectable non-radioactively labeled oligonucleotide or polynucleotide comprises one or more detectable non-radioactively modified or labeled nucleotides or detectable non-radioactively modified or labeled nucleotide analogs, which nucleotide analogs can be attached to or coupled to or incorporated into DNA or RNA, and (ii) a sample that may contain said nucleic acid of interest; forming in liquid phase, hybrids comprising said detectable non-radioactively labeled oligonucleotide or polynucleotide specifically hybridized with said nucleic acid of interest; and detecting hybrids non-radioactively to detect the presence of said nucleic acid of interest. - View Dependent Claims (190, 191)
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Specification