METHODS OF NONSPECIFIC TARGET CAPTURE OF NUCLEIC ACIDS
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.
1 . A method for isolating a target DNA from a sample, said method comprising:
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′; and
3) separating the support from the solution phase of the reaction mixture, thereby isolating the target DNA from other sample components.
2 . The method of claim 1 , wherein the sample contains cells and is treated before the mixing step to release intracellular components into the solution phase.
3 . The method of claim 2 , wherein said treatment comprises treating the sample with a solution containing a detergent.
4 . The method of claim 2 , wherein the sample comprises peripheral blood.
5 . The method of claim 1 , wherein the poly-(k) sequence comprises G and U nucleotides.
6 . The method of claim 5 , wherein the poly-(k) sequence comprises 2′-O-methyl substituted RNA bases, or wherein the nonrandom repeating (GU) sequence comprises 2′-O-methyl substituted RNA bases.
7 . The method of claim 1 , wherein the poly-(k) sequence is a poly-(k) 18 , poly-(k) 24 , or poly-(k) 25 sequence.
8 . The method of claim 1 , wherein the SBP and SBP′ are non-nucleic acid moieties.
9 . The method of claim 1 , wherein the SBP and SBP′ are substantially complementary nucleic acid sequences.
10 . The method of claim 9 , wherein the SBP is situated 3′ to the poly-(k) sequence.
11 . The method of claim 9 , wherein the SBP is a dT 3 dA 30 or dA 30 sequence.
12 . The method of claim 1 , wherein the solution phase comprises a detergent.
13 . The method of claim 1 , wherein the solution phase comprises lithium lauryl sulfate and lithium hydroxide.
14 . The method of claim 1 , further comprising detecting the presence of the target DNA isolated from other sample components, amplifying in vitro a sequence contained in the target DNA isolated from other sample components, or determining a sequence contained the target DNA isolated from other sample components.
15 . A method for isolating a target DNA from a peripheral blood sample, said method comprising:
1) treating a peripheral blood sample containing the target DNA to release intracellular components into a solution phase;
2) mixing the treated sample containing the target DNA in the solution phase with a non-specific capture probe, wherein said non-specific capture probe comprises
(i) a randomized poly-(k) 18 sequence comprising G and U nucleotides with 2′-methoxy substituted RNA bases, and
(ii) a first specific binding partner (SBP), wherein said SBP is capable of specifically binding a second specific binding partner (SBP′);
3) incubating a reaction mixture containing (a) the SBP′ immobilized on a support and (b) the mixture of the non-specific capture probe and the treated 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′; and
4) separating the support from the solution phase of the reaction mixture, thereby isolating the target DNA from other peripheral blood sample components.
16 . A method for isolating a target DNA from a sample, said method comprising:
1) mixing a sample containing the target DNA in a solution phase with a non-specific capture probe, wherein said sample is provided as a lysate made from cells comprising the target DNA, and 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′);
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′; and
3) separating the support from the solution phase of the reaction mixture, thereby isolating the target DNA from other sample components.
17 . The method of claim 16 , wherein the sample comprises peripheral blood.
18 . The method of claim 16 , wherein the poly-(k) sequence comprises G and U nucleotides.
19 . The method of claim 18 , wherein the poly-(k) sequence comprises 2′-O-methyl substituted RNA bases.
20 . The method of claim 16 , wherein the poly-(k) sequence is a poly-(k) 18 , poly - (k) 24 , or poly-(k) 25 sequence.
21 . The method of claim 16 , wherein the SBP and SBP′ are non-nucleic acid moieties.
22 . The method of claim 16 , wherein the SBP and SBP′ are substantially complementary nucleic acid sequences.
23 . The method of claim 22 , wherein the SBP is situated 3′ to the poly-(k) sequence.
24 . The method of claim 22 , wherein the SBP is a dT 3 dA 30 or dA 30 sequence.
25 . The method of claim 16 , wherein the solution phase comprises a detergent.
26 . The method of claim 16 , further comprising detecting the presence of the target DNA isolated from other sample components, amplifying in vitro a sequence contained in the target DNA isolated from other sample components, or determining a sequence contained the target DNA isolated from other sample components.
27 . A reaction mixture for isolating a target DNA from a sample, said reaction mixture comprising:
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′); and
c) the SBP′ immobilized on a support.
28 . The reaction mixture of claim 27 , wherein the sample is derived from cells that have been treated to release intracellular components into the solution phase.
29 . The reaction mixture of claim 28 , wherein the solution phase comprises a detergent used in said treatment.
30 . The reaction mixture of claim 28 , wherein the sample comprises peripheral blood.
31 . The reaction mixture of claim 27 , wherein the poly-(k) sequence comprises G and U nucleotides.
32 . The reaction mixture of claim 31 , wherein the poly-(k) sequence comprises 2′-O-methyl substituted RNA bases.
33 . The reaction mixture of claim 27 , wherein the poly-(k) sequence is a poly-(k) 18 , poly-(k) 24 , or poly-(k) 25 sequence.
34 . The reaction mixture of claim 27 , wherein the SBP and SBP′ are non-nucleic acid moieties.
35 . The reaction mixture of claim 27 , wherein the SBP and SBP′ are substantially complementary nucleic acid sequences.
36 . The reaction mixture of claim 35 , wherein the SBP is situated 3′ to the poly-(k) sequence.
37 . The reaction mixture of claim 35 , wherein the SBP is a dT 3 dA 30 or dA 30 sequence.
38 . The reaction mixture of claim 27 , wherein the solution phase comprises a detergent.
39 . The reaction mixture of claim 27 , wherein the solution phase comprises lithium lauryl sulfate and lithium hydroxide.