Methods of nonspecific target capture of nucleic acids
First Claim
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1. A method for isolating nucleic acid from a sample, comprising the steps of:
- a. mixing a sample containing nucleic acids with a non-specific capture probe comprising a first region that is a randomized poly(k) sequence at a length of 18 residues comprising G and T nucleotides or G and U nucleotides attached to a second region that is a first specific binding partner (SBP);
b. incubating a reaction mixture containing a support and the mixture of nucleic acids and non-specific capture probe in conditions that allow hybridization of the first region with all or a portion of the nucleic acids in the sample and that allow for hybridization of the SBP with a second specific binding partner (SBP′
) immobilized to the support, thereby forming a hybridization complex; and
c. separating the support from the solution phase of the reaction mixture, thereby isolating the nucleic acids away from other components in the sample.
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Abstract
Methods for capturing a target nucleic acid from a sample by using a capture probe that binds nonspecifically to the target nucleic acid and binds specifically to an immobilized probe via a specific binding pair that has one member on the capture probe and one member on the immobilized probe are disclosed. Compositions that include a capture probe that binds nonspecifically to a target nucleic acid and specifically to an immobilized probe via binding of members of a specific binding pair in a solution phase of a reaction mixture are disclosed.
41 Citations
53 Claims
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1. A method for isolating nucleic acid from a sample, comprising the steps of:
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a. mixing a sample containing nucleic acids with a non-specific capture probe comprising a first region that is a randomized poly(k) sequence at a length of 18 residues comprising G and T nucleotides or G and U nucleotides attached to a second region that is a first specific binding partner (SBP); b. incubating a reaction mixture containing a support and the mixture of nucleic acids and non-specific capture probe in conditions that allow hybridization of the first region with all or a portion of the nucleic acids in the sample and that allow for hybridization of the SBP with a second specific binding partner (SBP′
) immobilized to the support, thereby forming a hybridization complex; andc. separating the support from the solution phase of the reaction mixture, thereby isolating the nucleic acids away from other components in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method for isolating nucleic acid from a sample, comprising the steps of:
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a. mixing a sample containing nucleic acids with a non-specific capture probe comprising a first region that is from 6 to 24 randomized poly(k) residues, and from 0 to 2 C9 linkers or from 0 to 1 5-nitroindole, and the first region is attached to a second region that is a specific binding partner (SBP); b. incubating the mixture of nucleic acids and non-specific capture probe in a target capture mixture under conditions that allow hybridization of the first region with all or a portion of the nucleic acids in the sample; and c. separating out of the solution phase all or a portion of the nucleic acids hybridized with a non-specific capture probe, thereby isolating the nucleic acids away from other components in the sample. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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27. A method for isolating nucleic acid from a sample, comprising the steps of:
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a. forming a target capture reaction mixture to include nucleic acids from a sample and a non-specific capture probe comprising a first region that is from 6 to 24 randomized poly(k) residues, and from 0 to 2 non-nucleotide linkers or from 0 to 1 nucleotide analogs, and the first region is attached to a second region that is a specific binding partner (SBP); b. incubating the mixture at step a for at least 1 minute and under conditions that allow hybridization of the first region with all or a portion of the nucleic acids in the sample; and c. separating out of the solution phase all or a portion of the nucleic acids hybridized with a non-specific capture probe, thereby isolating the nucleic acids away from other components in the sample. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34)
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35. A method for isolating a target DNA from a sample, said method comprising:
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1) mixing a sample containing the target DNA in a solution phase with a non-specific capture probe, wherein said non-specific capture probe comprises (i) a randomized poly-(k) sequence that is at least 12 residues in length and comprising G and T nucleotides or G and U nucleotides, or a nonrandom repeating (GU) sequence that is at least 12 residues in length, and (ii) a first specific binding partner (SBP), wherein said SBP is capable of specifically binding a second specific binding partner (SBP′
);2) incubating a reaction mixture containing (a) the SBP′
immobilized on a support and (b) the mixture of the non-specific capture probe and the sample containing the target DNA, wherein said incubating is under conditions that allow hybridization of the capture probe'"'"'s poly-(k) sequence with the target DNA and that allow specific binding of the SBP to the SBP′
; and3) separating the support from the solution phase of the reaction mixture, thereby isolating the target DNA from other sample components. - View Dependent Claims (36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)
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49. A reaction mixture for isolating a target DNA from a sample, said reaction mixture comprising:
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a) a sample containing the target DNA in a solution phase; b) a non-specific capture probe, wherein said non-specific capture probe comprises (i) a randomized poly-(k) sequence that is at least 12 residues in length and comprising G and T nucleotides or G and U nucleotides, and (ii) a first specific binding partner (SBP), wherein said SBP is capable of specifically binding a second specific binding partner (SBP′
); andc) the SBP′
immobilized on a support. - View Dependent Claims (50, 51, 52, 53)
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Specification