Compositions and methods for detecting and quantifying gene expression
First Claim
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1. A microarray comprising a surface silanized with a silane in toluene in the absence of acetone or an alcohol, and a target molecule, wherein the target molecule is attached to the surface via the silane.
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Abstract
Compositions and methods for improving detection sensitivity in nucleic acid microarray analysis are disclosed, including methods of purifying nucleic acids, methods of synthesizing fluorescent DNA probes, methods of hybridization, and methods of activating a substrate for target molecule attachment are disclosed.
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Citations
104 Claims
- 1. A microarray comprising a surface silanized with a silane in toluene in the absence of acetone or an alcohol, and a target molecule, wherein the target molecule is attached to the surface via the silane.
- 2. A microarray comprising a surface silanized with a silane in toluene in the absence of acetone or an alcohol, a linker, and a target molecule, wherein the target molecule is attached to the surface via the linker.
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25. A microarray prepared by a method comprising:
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(a) providing a multifunctional linker reagent comprising two or more reactive groups capable of reacting with a functional group on a surface of a microarray substrate and capable of reacting with a target molecule;
(b) activating the substrate surface for immobilizing the target molecule, by silanizing the surface with a silane in toluene in the absence of acetone or an alcohol, wherein the silane comprises a functionality reactive with the multifunctional linker reagent, and wherein the activating further comprises immobilizing the multifunctional linker reagent on the silanized surface by attaching the multifunctional linker reagent to the silane via a first reactive group of the linker reagent and a reactive group of the silane;
(c) providing a solution comprising a target molecule having one or more functional groups reactive with a second reactive group of the immobilized multifunctional linker reagent;
(d) attaching the target molecule to the substrate surface by contacting the target molecule with the activated substrate surface under conditions that promote attachment of the target molecule to the immobilized multifunctional linker reagent. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)
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- 41. An activated slide comprising a substrate surface comprising a silane attached thereto, wherein the silanizing was in toluene, in the absence of acetone or an alcohol, and wherein the attached silane comprises at least one reactive functionality that is capable of reacting with a compound to immobilize the compound on the substrate surface.
- 52. A method of activating a glass slide for immobilizing a target molecule, the method comprising silanizing the slide with a silane in toluene in the absence of acetone or an alcohol, wherein the silane is an alkyl silane and the alkyl moiety is selected from the group consisting of an ethyl-, a propyl-, a butyl-, a pentyl-, a hexyl-, a heptyl-, an octyl-, a nonyl-, and a decylalkyl moiety, and the reactive functionality of the silane is selected from the group consisting of an amine, a hydroxyl moiety, an epoxide, a thiol, and a halide, and the reactive functionality is covalently linked to the alkyl moiety.
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55. A method of preparing a microarray, the method comprising:
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(a) providing an activated slide comprising a substrate surface comprising a silane attached thereto, wherein the silanizing was in toluene, in the absence of acetone or an alcohol, and wherein the attached silane comprises at least one reactive functionality that is capable of reacting to immobilize a target molecule on the substrate surface;
(b) reacting the activated slide surface with the target molecule under conditions to immobilize the target molecule, wherein the target molecule is selected from the group consisting of a nucleic acid, a polynucleotide, RNA, single stranded DNA, double stranded DNA, an oligonucleotide, a peptide nucleic acid (PNA), a polypeptide, a protein, an antibody, a receptor, and a ligand. - View Dependent Claims (56, 57, 58, 59, 60, 61, 62, 63, 64)
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65. A method of preparing a detectably labeled sDNA probe capable of forming a detectable complex with a target molecule immobilized on a microarray surface, the method comprising:
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(a) isolating an amount of total cellular RNA from a biological sample;
(b) synthesizing a mixture of detectably labeled sDNA probes, wherein the synthesis of sDNA comprises synthesizing first strand cDNA from the isolated RNA of step (a), synthesizing second strand cDNA using Klenow fragment of DNA polymerase I and the first strand cDNA as template, synthesizing cRNA using the double stranded cDNA as template; and
synthesizing sDNA using reverse transcriptase in the presence of detectably labeled deoxyribonucleotide using the cRNA as a template;
(c) isolating the labeled sDNA probes. - View Dependent Claims (66, 67, 68, 69, 77, 78, 79, 80, 81, 82, 83)
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70. A method of preparing a detectably labeled cDNA probe capable of forming a detectable complex with a target molecule immobilized on a microarray surface, the method comprising:
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(a) isolating an amount of total cellular RNA from a biological sample;
(b) synthesizing a mixture of detectably labeled cDNA probes, wherein the synthesis of cDNA comprises synthesizing first strand cDNA from the isolated RNA of step (a) in the presence of detectably labeled deoxynucleotide;
(c) isolating the labeled sDNA probes. - View Dependent Claims (75, 76)
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71. A method of preparing a detectably labeled sDNA probe capable of forming a detectable complex with a target molecule immobilized on a microarray surface, the method comprising:
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(a) isolating an amount of total cellular RNA from a biological sample;
(b) synthesizing a mixture of detectably labeled sDNA probes, wherein the synthesis of sDNA comprises synthesizing a biotin-attached first strand cDNA from the isolated RNA of step (a);
synthesizing second strand DNA (sDNA) using Klenow fragment of DNA polymerase I and the first strand cDNA as template in the presence of detectably labeled deoxynucleotides;
(c) contacting the biotin-attached first strand cDNA with streptavidin and removing the biotin-first strand cDNA/streptavidn complex from the labeled sDNA; and
(c) isolating the labeled sDNA probes. - View Dependent Claims (74)
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72. A method of preparing a detectably labeled cRNA probe capable of forming a detectable complex with a target molecule immobilized on a microarray surface, the method comprising:
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(a) isolating an amount of total cellular RNA from a biological sample;
(b) synthesizing a mixture of detectably labeled CRNA probes, wherein the synthesis of cRNA comprises synthesizing first strand cDNA from the isolated RNA of step (a), synthesizing second strand CDNA using Klenow fragment of DNA polymerase I and the first strand cDNA as template, synthesizing cRNA using the double stranded cDNA as template in the presence of detectably labeled ribonucleotides; and
(c) isolating the labeled sDNA probes. - View Dependent Claims (73)
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93. A method of hybridizing a detectable polynucleotide probe to a target polynucleotide on a support surface, the method comprising:
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(a) contacting the probe with denatured target polynucleotide on the support surface in a hybridization solution comprising DMSO or formamide or both, and in the absence of detergent; and
(b) detecting formation of a complex between the target polynucleotide and the detectably labeled polynucleotide probe. - View Dependent Claims (94, 95)
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Specification