Arrayed biomolecules and their use in sequencing
First Claim
1. A device comprising a high density array of molecules capable of interrogation and immobilised on a solid planar surface, wherein the array allows the molecules to be individually resolved by optical microscopy, and wherein each molecule is immobilised by covalent bonding to the surface, other than at that part of each molecule a can be interrogated.
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Abstract
A device comprising an array of molecules immobilised on a solid surface is disclosed, wherein the array has a surface density which allows each molecule to be individually resolved, e.g. by optical microscopy. Therefore, the arrays of the present invention consist of single molecules are more spatially distinct than the arrays of the prior art.
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Citations
42 Claims
- 1. A device comprising a high density array of molecules capable of interrogation and immobilised on a solid planar surface, wherein the array allows the molecules to be individually resolved by optical microscopy, and wherein each molecule is immobilised by covalent bonding to the surface, other than at that part of each molecule a can be interrogated.
- 13. A device comprising a high density array of relatively short molecules and relatively long polynucleotides immobilised on the surface of a solid support, wherein the polynucleotides are at a density that permits individual resolution of those parts thereof that extend beyond the relatively short molecules.
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15. A device comprising an array of polynucleotide molecules immobilised on a solid surface, wherein each molecule comprises a polynucleotide duplex liked via a covalent bond to form a hairpin loop structure, one end of which comprises a target polynucleotide, and the array has a surface density which allows the target polynucleotides to be individually resolved.
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16. A method for producing a device comprising a high density array of molecules capable of interrogation an immobilised on a solid planar surface, wherein the array allows the molecules to be individually resolved by optical microscopy, and wherein each molecule is immobilised by covalent bonding to the surface, other than at that part of each molecule that can be interrogated, the method comprising dispensing a solution comprising a mixture of molecules onto a solid surface under conditions that permit immobilisation and that minimise aggregation of the molecules in solution.
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17. A method for producing a device comprising a high density array of polynucleotide molecules capable of interrogation and immobilised on a solid planar surface, wherein the array allows the polynucleotide molecules to be individually resolved by optical microscopy, and wherein each polynucleotide molecule is immobilised by covalent bonding to the surface, other than at that part of each polynucleotide molecule that can be interrogated, the method comprising (i) immobilising primer polynucleotides at discrete sites on the surface of a solid support, and (ii) contacting the immobilised primers with target polynucleotides under hybridising conditions
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18. A method for producing a device comprising a high density array of polynucleotide molecules capable of interrogation and immobilised on a solid planar surface, wherein the array allows the polynucleotide molecules to be individually resolved by optical microscopy, and wherein each polynucleotide molecule is immobilised by covalent bonding to the surface, other than at that part of each molecule that can be interrogated, the method comprising (i) immobilising first polynucleotides at discrete sites on the surface of a solid support, and hybridising thereto second polynucleotides which form single-stranded overhangs, (ii) contacting the product of step (i) with target polynucleotides under hybridising conditions;
- (iii) ligating the target polynucleotides to the first polynucleotides with a DNA ligase, and, optionally, (iv) removing the second polynucleotides
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19. A method for the preparation of a device comprising an array of polynucleotide molecules immobilised on a solid surface, wherein each molecule comprises a polynucleotide duplex linked via a covalent bond to form a hairpin loop structure, one end of which comprises a target polynucleotide, and the array has a surface density which allows the target polynucleotides to be individually resolved, the method comprising ligating a target polynucleotide to the 5′
- end of a first molecule capable of forming said duplex, and immobilising the first molecule to the solid surface either before or after ligation.
- View Dependent Claims (20, 21, 22, 23)
- 24. A method for forming a spatially addressable array, which comprises determining the sequences of a plurality of polynucleotide molecules immobilised on a device comprising a high density array of molecules capable of interrogation and immobilised on a solid planar surface, wherein the array allows the molecules to be individually resolved by optical microscopy, and wherein each molecule is immobilised by covalent bonding to the surface, other than at that part of each molecule that can be interrogated
- 27. A method for characterising a plurality of first molecules, comprising contacting, under suitable conditions, a spatially addressed array of second molecules with the first molecules, and detecting a binding event, wherein the array comprises a high density array of molecules capable of interrogation and immobilised on a solid planar surface, wherein the array allows the molecules to be individually resolved by optical microscopy, and wherein each molecule is immobilised by covalent bonding to the surface, other than at that part of each molecule that can be interrogated
- 31. A method for characterising an organism, comprising the steps of contacting a defined array of polynucleotide molecules immobilised on a solid support with a plurality of fragments of the organisms genomic DNA, under hybridising condiions, and detecting any hybridisation events, to obtain a distinct hybridisation pattern, wherein the array is comprising a high density array of molecules capable of interrogation and immobilised on a solid planar surface, wherein the array allows the molecules to be individually resolved by optical microscopy, and wherein each molecule is immobilised by covalent bond to the surface, other than at that part of each molecule that can be interrogated
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35. A method for determining a single nucleotide polymorphism present in a genome, comprising (i) immobilising fragments of said genome onto the surface of a solid support to form an array of polynucleotide molecules capable of interrogation, wherein the array allows the molecules to be individually resolved by optical microscopy, and wherein each molecule is immobilised by covalent bonding to the surface, other than at that part of each molecule that can be interrogated;
- (ii) identifying nucleotides at selected positions in the genome, and (iii) comparing the results of step (ii) with a known consensus sequence, and identifying any differences between the consensus sequence and said genome.
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36. A method for determining a single nucleotide polymorphism present in a genome, comprising (i) immobilising fragments of sad genome onto the surface of a solid support to form an array of polynucleotide molecules capable of interrogation, wherein the array allows the molecules to be individually resolved by optical microscopy, and wherein each molecule is immobilised by covalent bonding to the surface, other than at that part of each molecule that can be interrogated;
- (ii) contacting the array with each of the bases A, T, G and C, under conditions that permit the polymerase reaction to proceed and thereby form sequences complementary to those in the array, (iii) determining the incorporation of a base at each of selected positions in the complementary sequences;
(iv) optionally repeating steps (ii) and (iii); and
(v) comparing the result of step (iii) with a known consensus sequence, and identifying any differences between the consensus sequence and said genome - View Dependent Claims (37)
- (ii) contacting the array with each of the bases A, T, G and C, under conditions that permit the polymerase reaction to proceed and thereby form sequences complementary to those in the array, (iii) determining the incorporation of a base at each of selected positions in the complementary sequences;
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38. Use of a device comprising a high density array of polynucleotide molecules capable of interrogation and immobilised on a solid planar surface, wherein the array allows the molecules to be individually resolved by optical microscopy, and wherein each molecule is immobilised by covalent bonding to the surface via the 5′
- 0 terminus, the 3′
terminus, or via an internal nucleotide, for the capture of a second polynucleotide molecule capable of hybridising with the arrayed polynucleotide, comprising bringing into contact with the device a sample containing or suspected of containing the second polynucleotide molecule, under hybridising conditions. - View Dependent Claims (39)
- 0 terminus, the 3′
- 40. Use of a device comprising a high density array of molecules capable of interrogation and immobilised on a solid planar surface, wherein the array allows the molecules to be individually resolved by optical microscopy, and wherein each molecule is immobilised by covalent bond to the surface, other than a that part of each molecule that can be interrogated, for monitoring an interaction with a single molecule, comprising resolving an arrayed molecule with an imaging device.
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42. Use of a device comprising an array of molecules immobilised on a solid surface, wherein each molecule comprises a polynucleotide duplex linked via a covalent bond to form a hairpin loop structure, one end of which comprises a target polynucleotide, and the array has a surface density which allows the target polynucleotides to be individually resolved, in an analysis procedure to determine the sequence of the target polynucleotide.
Specification