pH dependent ion exchange matrix and method of use in the isolation of nucleic acids
First Claim
1. A pH dependent ion exchange matrix for isolating a target nucleic acid, comprising:
- a silica magnetic particle; and
a plurality of first ion exchange ligands, each first ion exchange ligand comprising;
an aromatic hydrocarbon ring, wherein at least one member of the ring is an amine with a pK of less than about 9;
a spacer covalently attached to the aromatic hydrocarbon ring, the spacer comprising a spacer alkyl chain of with an amine terminus, and an acidic moiety covalently attached to the spacer alkyl chain; and
a linker comprising a linker alkyl chain covalently attached to the silica magnetic particle through a silica residue at a first end of the linker alkyl chain and covalently attached to the amine terminus of the spacer at a second end of the linker alkyl chain;
wherein the matrix has a capacity to adsorb to a target nucleic acid at a first pH, and to release the target nucleic acid at a desorption pH which is higher than the first pH.
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Abstract
pH dependent ion exchange matrices are provided, with methods for making such matrices, and methods for using such matrices to isolate a target nucleic acid, such as plasmid DNA, chromosomal DNA, or RNA from contaminants, including proteins, lipids, cellular debris, or other nucleic acids. Each pH dependent ion exchange matrix of this invention comprises at least two different ion exchange functional groups, one of which is capable of acting as an anion exchanger at a first pH, and the other of which is capable of acting as a cation exchanger at a second, higher pH. The matrix has an overall neutral charge in a pH range between the first and second pH. The pH dependent ion exchange matrices of the present invention are designed to bind to the target nucleic acid at a pH wherein the overall charge of the matrix is positive, and to release the target nucleic acid as the pH of the surrounding solution is increased. The target nucleic acid can be released from the pH dependent matrix in little or no salt and at about a neutral pH. The matrices and methods of this invention enable one to isolate a target nucleic acid in very few steps, without the use of hazardous chemicals. Target nucleic acids isolated using the pH dependent ion exchange matrices according to the present invention can be used immediately without further extraction or isolation.
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Citations
70 Claims
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1. A pH dependent ion exchange matrix for isolating a target nucleic acid, comprising:
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a silica magnetic particle; and
a plurality of first ion exchange ligands, each first ion exchange ligand comprising;
an aromatic hydrocarbon ring, wherein at least one member of the ring is an amine with a pK of less than about 9;
a spacer covalently attached to the aromatic hydrocarbon ring, the spacer comprising a spacer alkyl chain of with an amine terminus, and an acidic moiety covalently attached to the spacer alkyl chain; and
a linker comprising a linker alkyl chain covalently attached to the silica magnetic particle through a silica residue at a first end of the linker alkyl chain and covalently attached to the amine terminus of the spacer at a second end of the linker alkyl chain;
wherein the matrix has a capacity to adsorb to a target nucleic acid at a first pH, and to release the target nucleic acid at a desorption pH which is higher than the first pH. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
glycidine and urea.
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13. The matrix of claim 1, wherein the matrix is an anion exchanger capable of exchanging with the target nucleic acid at the first pH, and the matrix was a net neutral or negative charge at the desorption pH is not.
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14. The matrix of claim 1, wherein the matrix can be reused through at least two cycles of adherence of the target nucleic acid to the matrix at the first pH and release from the matrix at the desorption pH.
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15. The matrix of claim 9, wherein the linker is urea.
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16. The matrix of claim 9, wherein the matrix is an anion exchanger capable of exchanging with the target nucleic acid at the first pH, neutral at a second pH which is higher than the first pH, and a cation exchanger at a third pH which is higher than the second pH.
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17. The matrix of claim 9, wherein the proportion of the plurality of first ion exchange ligands and the plurality of second ion exchange ligands covalently attached to the solid phase is designed to ensure that when the matrix comes into contact with a solution comprising a target nucleic acid at the first pH, the matrix preferentially binds to the target nucleic acid.
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18. The matrix of claim 9, wherein the matrix can be reused through at least two cycles of adherence of the target nucleic acid to the matrix at the first pH and release from the matrix at the desorption pH.
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19. A multimodal pH dependent ion exchange matrix, comprising:
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a solid support;
a plurality of first ion exchange ligands, each first ion exchange ligand comprising;
a cap comprising an amine with a pK of less than about 9;
a spacer covalently attached to the cap, the spacer comprising a spacer alkyl chain with an amine terminus; and
a linker comprising a linker alkyl chain covalently attached to the solid support at a first end of the linker alkyl chain and covalently attached to the amine terminus of the spacer at a second end of the linker alkyl chain;
a plurality of second ion exchange ligands, each second ion exchange ligand comprising;
a second alkyl chain covalently attached to the solid support at a first end of the second alkyl chain; and
a second acidic moiety covalently attached to a second end of the second alkyl chain, wherein the matrix has a capacity to adsorb to a target nucleic acid at a first pH, and to release the target nucleic acid at a desorption pH which is higher than the first pH. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
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37. A method of isolating a target nucleic acid using a multimodal pH dependent ion exchange matrix, comprising the steps of:
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(a) providing a multimodal pH dependent ion exchange matrix comprising;
a solid support;
a plurality of first ion exchange ligands, each first ion exchange ligand comprising;
a cap comprising an amine with a pK of less than 9, wherein the amine is selected from the group consisting of a primary, a secondary, and a tertiary amine;
a spacer covalently attached to the cap, the spacer comprising a spacer alkyl chain with an amine terminus; and
a linker comprising a linker alkyl chain covalently attached to the solid support at a first end of the linker alkyl chain and covalently attached to the amine terminus of the spacer at a second end of the linker alkyl chain;
a plurality of second ion exchange ligands comprising;
a second alkyl chain covalently attached to the solid support at a first end of the second alkyl chain; and
a second acidic moiety covalently attached to a second end of the second alkyl chain, wherein the matrix has a capacity to adsorb to a target nucleic acid at a first pH, and to release the target nucleic acid at a desorption pH which is higher than the first pH, (b) providing a mixture comprising the target nucleic acid;
(c) combining the mixture and the matrix and incubate at the first pH until the nucleic acid adsorbs to the matrix, forming a complex;
(d) separating the complex from the mixture; and
(e) combining the complex with an elution solution at the desorption pH. - View Dependent Claims (38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57)
histamine via epoxide, histidine via epoxide, histidine via uiea. histidine via sulfhydryl, pyridyl alanine, pyridyl cysteine.
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56. The method of claim 54, wherein the target nucleic acid is plasmid DNA.
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57. The method of claim 54, wherein the target nucleic acid is genomic DNA.
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58. A method of making a pH dependent ion exchange matrix, comprising the steps of:
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(a) providing a solid phase;
(b) providing a linker comprising an alkyl chain having a first end and a second end;
(c) combining the silica based solid phase and the linker under conditions where a covalent bond is formed between the solid phase and the first end of the linker alkyl chain, thereby producing a linker-modified solid phase;
(d) providing an acidic aromatic amine comprising;
an aromatic hydrocarbon ring, wherein at least one member of the ring is an amine;
a spacer which is covalently attached to the aromatic hydrocarbon, wherein the spacer comprises a spacer alkyl chain with an amino terminus; and
an acidic substituent which is covalently attached to the spacer alkyl chain; and
(e) combining the linker-modified solid phase with the acidic aromatic amine under conditions where a covalent bond is formed between the amino terminus of the spacer alkyl chain of the acidic aromatic amine and the second end of the linker. - View Dependent Claims (59, 60, 61, 62, 63, 64, 65, 66, 67, 68)
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69. A method of making a bimodal pH dependent ion exchange matrix, comprising the steps of:
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(a) providing a solid support;
(b) providing a first ion exchange ligand comprising;
a cap comprising an amine with a pK of less than about 9, wherein the amine is selected from the group consisting of a primary, a secondary, or a teritary amine;
a spacer covalently attached to the cap, the spacer comprising a spacer alkyl chain and with an amine terminus; and
a linker comprising a linker alkyl chain having a first end and a second end, wherein the second end is covalently attached to the amine terminus of the spacer;
(c) combining the solid phase and the first ion exchange ligand under conditions where a covalent bond is formed between solid phase and the first end of the linker alkyl chain;
(d) providing a second ion exchange ligand, comprising a second alkyl chain and an acidic residue covalently attached thereto, wherein the acidic residue has a protective group covalently attached thereto;
(e) combining the solid phase with the first ion exchange ligand attached thereto with a second ligand under conditions which promote formation of a covalent bond between the protected second ion exchange ligand and the solid phase; and
(f) deprotecting the acidic residue of the second anion exchange ligand by removing the protective group therefrom. - View Dependent Claims (70)
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Specification