Methods of immobilizing ligands on solid supports and apparatus and methods of use therefor
First Claim
Patent Images
1. A method of immobilizing an affinity ligand on a solid support comprising:
- providing a solid support comprising an immobilized thiol group;
contacting the thiol group with a nucleic acid comprising an acrylamide functional group; and
forming a covalent bond between the two groups, thereby immobilizing the ligand on the solid support.
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Abstract
A method is provided for immobilizing a ligand, e.g., a nucleic acid, on a solid support. The method includes providing a solid support containing an immobilized latent thiol group, activating the thiol group, contacting the activated thiol group with a nucleic acid comprising an acrylamide functional group, and forming a covalent bond between the two groups, thereby immobilizing the nucleic acid to the solid support. Kits containing the solid supports and method of utilizing the solid supports are also provided.
27 Citations
71 Claims
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1. A method of immobilizing an affinity ligand on a solid support comprising:
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providing a solid support comprising an immobilized thiol group;
contacting the thiol group with a nucleic acid comprising an acrylamide functional group; and
forming a covalent bond between the two groups, thereby immobilizing the ligand on the solid support. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method of immobilizing an affinity ligand on a solid support comprising the steps of:
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providing a solid support comprising immobilized latent thiol groups;
activating the latent thiol groups; and
reacting the activated thiol groups with an affinity ligand having at least one acrylamide functional group, thereby immobilizing an affinity ligand on a solid support. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 21, 22, 23, 24, 25, 26, 27, 28, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70)
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18. A method of immobilizing an affinity ligand on microarray comprising the steps of:
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providing a solid support comprising immobilized latent thiol groups;
activating the latent thiol groups; and
reacting the activated thiol groups with an affinity ligand having at least one α
,β
unsaturated carbonyl functional group, thereby immobilizing an affinity ligand on a solid support. - View Dependent Claims (19, 20)
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29. A method of preparing a solid support having immobilized thiol groups, comprising the steps of:
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contacting a glass solid support with a silane compound represented by the following structural formula;
wherein;
X is a halogen; and
R1, R2, and R3, are each, independently, a halogen, an alkyl group, an alkenyl group or a group having at least one α
,β
-unsaturated carbonyl, provided that at least one of R1, R2, or R3 is an alkenyl group or a group having at least one α
,β
-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface;
contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing free radical initiator, disulfide bisacrylamide, and optionally an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula;
wherein;
n and m are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups; and
contacting the latent thiol groups with a disulfide reducing agent, thereby forming a solid support having immobilized thiol groups. - View Dependent Claims (30, 31, 32, 33)
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34. A method of forming an array of nucleic acids immobilized on a solid support comprising:
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forming an amine-derivatized region on the support;
treating the amine-derivatized region with a thiolating agent such that latent thiol groups immobilized on the support are formed;
activating the latent thiol groups;
contacting the activated thiol groups with a plurality of nucleic acids comprising acrylamide functional groups; and
forming a covalent bond between the two groups, thereby forming an array of nucleic acids immobilized on the solid support. - View Dependent Claims (35, 36, 37, 38, 39)
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- 40. A kit for attaching nucleic acids to a solid support comprising a solid support component comprising a plurality of immobilized latent thiol groups and instructions for activating the thiol groups to form covalent bonds with nucleic acids comprising acrylamide functional groups.
- 42. A kit for attaching nucleic acids to a solid support comprising a solid support component comprising a plurality of immobilized thiol groups and nucleic acids comprising acrylamide functional groups.
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45. A method for detecting or separating target nucleic acids from other components contained in a sample comprising:
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providing a solid support comprising a plurality of immobilized nucleic acids comprising nucleotide sequences complementary to a subsequence of the nucleotide sequence of the target nucleic acid, wherein the nucleic acids are immobilized by a covalent bond formed between a thiol group immobilized on the solid support and an acrylamide functional group contained on the nucleic acid;
contacting the immobilized nucleic acid with the sample; and
hybridizing target nucleic acids to immobilized nucleic acids with complementary subsequences, thereby detecting or separating target nucleic acids from other components contained in the sample. - View Dependent Claims (46, 47)
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48. A method for detecting or separating target nucleic acids from other components contained in a sample comprising:
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providing a solid support comprising a plurality of immobilized thiol groups;
contacting the thiol groups with a plurality of nucleic acids comprising nucleotide sequences complementary to a subsequence of the nucleotide sequence of the target nucleic acid and acrylamide functional groups;
forming a covalent bond between the thiol and acrylamide functional groups, thereby immobilizing the nucleic acids on the solid support;
contacting the immobilized nucleic acids with the sample; and
hybridizing target nucleic acids to immobilized nucleic acids with complementary subsequences, thereby detecting or separating target nucleic acids from other components contained in the sample. - View Dependent Claims (49, 50)
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71. A method of making a solid support having immobilized thiol groups, comprising the steps of:
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contacting a glass solid support with a silane compound represented by the following structural formula;
wherein;
X is a halogen; and
R1, R2 and R3 are each, independently, a halogen, an alkyl group, an alkenyl group or a group having at least one α
,β
-unsaturated carbonyl, provided that at least one of RI, R, or R, is an alkenyl group or group having at least one α
,β
-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface;
contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing free radical initiator, a disulfide bisacrylamide and optionally containing an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula;
wherein;
n and m are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups; and
contacting the latent thiol groups with a disulfide reducing agent, thereby forming a solid support having immobilized thiol groups.
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Specification