Method 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 reaction mixture for isolating a target nucleic acid from a sample, said reaction mixture comprising:
(a) a sample derived from cells that have been treated to release intracellular components, including the target nucleic acid, into a solution phase;
(b) 15-40 pmol of a non-specific capture probe, wherein the non-specific capture probe comprises a first specific binding partner (SBP) and an oligonucleotide sequence wherein the oligonucleotide sequence is selected from the group consisting of:
(i) two random poly-(k) sequences synthesized by random incorporation of G and T/U nucleotides separated by a spacer, wherein the total length of the two random poly-(k) sequences is at least 12 residues and wherein the spacer is selected from the group consisting of:
(a) a non-random sequence represented by a formula N 1-10 , wherein N is A/U/T/G/C;
(b) a non-random sequence consisting of 10 or fewer inosine;
(c) a non-random sequence consisting of 10 or fewer 5-nitroindole; and
(d) a non-nucleotide spacer consisting of one or two C9 spacers;
(ii) a plurality of random poly-(k) sequences synthesized by random incorporation of G and T/U nucleotides wherein the total length of the plurality of random poly-(k) sequences is at least 12 residues and wherein the random poly-(k) sequences are each separated by a spacer selected from the group consisting of:
(a) non-random sequence represented by a formula N 1-10 , wherein N is A/U/T/G/C;
(b) a non-random sequence consisting of 10 or fewer inosine;
(c) a non-random sequence consisting of 10 or fewer 5-nitroindole; or
(d) a non-nucleotide spacer consisting of one or two C9 spacers;
(iii) a non-random poly-GU sequence made up of at least six GU units; and
(iv) a random poly-(k) sequence synthesized by random incorporation of G and T/U nucleotides wherein the random poly-(k) is at least 12 residues in length;
wherein the SBP comprises a homopolymeric nucleic acid sequence at least 14 nucleotides in length, a dT 0-3 dA 30 , or a non-nucleic acid moiety; and
(c) a second specific binding partner (SBP′), wherein the SBP′ is immobilized to a support, and wherein the SBP specifically binds the SBP′.
2. The reaction mixture of claim 1 , wherein the solution phase comprises a detergent, lithium lauryl sulfate, or lithium hydroxide.
3. The reaction mixture of claim 1 , wherein the sample comprises peripheral blood.
4. The reaction mixture of claim 1 , wherein the oligonucleotide sequence contains one or more standard RNA bases and linkages, standard DNA bases and linkages, RNA bases with 2′ modified linkages, or a combination thereof.
5. The reaction mixture of claim 4 , wherein the oligonucleotide sequence comprises one or more 2′-O-methyl substituted RNA bases.
6. The reaction mixture of claim 1 , wherein the SBP and SBP′ are (a) non-nucleic acid moieties, or (b) substantially complementary nucleic acid sequences.
7. The reaction mixture of claim 6 , wherein the non-nucleic acid moieties are selected from the group consisting of: (a) a receptor and ligand pair, (b) an enzyme and substrate pair, (c) an enzyme and cofactor pair, (d) an enzyme and coenzyme pair, (e) an antibody and antigen pair, (f) an antibody fragment and antigen pair, (g) a sugar and lectin pair, (h) a ligand and chelating agent pair, (i) biotin and avidin, (j) biotin and streptavidin, and (k) nickel and histidine.
8. The reaction mixture of claim 1 , wherein the SBP (a) is situated 3′ to the oligonucleotide sequence, or (b) is a dT 3 dA 30 or dA 30 sequence, or (c) is (a) and (b).
9. The reaction mixture of claim 1 , wherein the oligonucleotide sequence is selected from the group consisting of:
(k)6-Ni5-(k)6 in which “Ni” stands for 5-nitroindole,
(k)6-C9-C9-(k)6 in which “C9” stands for a C9 spacer,
(k)6-C9-(k)6-C9-(k)6 in which “C9” stands for a C9 spacer,
L(k)4-d(k)3-L(k)4-d(k)3-L(k)4,
L(k)3-d(k)3-L(k)3-d(k)3-L(k)3-d(k)3,
L(k)2-d(k)4-L(k)2-d(k)4-L(k)2-d(k)4, and
L(k)4-d(k)3-L(k)4-d(k)3-L(k)4-d(k)3-L(k) 4,
wherein d designates that the sequence has standard DNA conformation and L designates that the sequence is in a locked nucleic acid (LNA) conformation.
10. The reaction mixture of claim 1 , wherein the oligonucleotide sequence is made up of two or more random poly-(k) sequences synthesized by random incorporation of G and T/U nucleotides separated by 1 to 2 C9 linkers or 1-5 5-nitroindole, and the SBP is dT 3 dA 30 , wherein each random poly-(k) sequence is 6-24 nucleotides in length.
11. The reaction mixture of claim 1 , wherein the SBP′ is linked to a magnetically attractable particle or a monodisperse paramagnetic bead.
12. A reaction mixture for isolating a target nucleic acid from a sample, the reaction mixture comprising:
a) a sample derived from cells that have been treated to release intracellular components, including the target nucleic acid, into a solution phase;
b) 15-40 pmol of a non-specific capture probe, wherein the non-specific capture probe comprises
(i) an oligonucleotide sequence that hybridizes nonspecifically to a target nucleic acid, wherein the oligonucleotide sequence comprises a randomized poly-(k) sequence synthesized by random incorporation of G and T nucleotides or G and U nucleotides, wherein the randomized poly-(k) sequence is 6 to 25 nucleotides in length and
(ii) a first specific binding partner (SBP) comprising a homopolymeric sequence; and
(c) a second specific binding partner (SBP′) wherein the SBP′ is immobilized to a support and contains a sequence that hybridizes specifically to the SBP.
13. The reaction mixture of claim 12 , wherein the solution phase comprises a detergent, lithium lauryl sulfate, or lithium hydroxide.
14. The reaction mixture of claim 12 , wherein the sample comprises peripheral blood.
15. The reaction mixture of claim 12 , wherein the oligonucleotide sequence contains one or more standard RNA bases and linkages, standard DNA bases and linkages, RNA bases with 2′ modified linkages, or a combination thereof.
16. The reaction mixture of claim 12 , wherein the oligonucleotide sequence contains a mixture of nucleotides in LNA conformation and standard DNA conformation.
17. The reaction mixture of claim 12 , wherein the randomized poly-(k) sequence is 12 to 25 nucleotides in length.
18. The reaction mixture of claim 12 , wherein the SBP comprises dT 3 dA 3 LA 1 dA 3 LA 1 dA 1 LA 1 dA 1 LA 1 dA 3 LA 1 dA 3 LA 1 dA 1 LA 1 dA 1 LA 2 dA 3 LA 1 dA 1 , wherein d designates that the sequence has a standard DNA conformation and L designates that the sequence is in a locked nucleic acid (LNA) conformation.
19. The reaction mixture of claim 12 , wherein the oligonucleotide sequence is selected from the group consisting of:
(k) 6 ,
(k) 12 ,
(k) 18 ,
(k) 24 ,
(k) 25 ,
L(k) 4 -d(k) 3 -L(k) 4 -d(k) 3 -L(k) 4 ,
L(k) 3 -d(k) 3 -L(k) 3 -d(k) 3 -L(k) 3 -d(k) 3 ,
L(k) 2 -d(k) 4 -L(k) 2 -d(k) 4 -L(k) 2 -d(k) 4 , and
L(k) 4 -d(k) 3 -L(k) 4 -d(k) 3 -L(k) 4 -d(k) 3 -L(k) 4 ,
wherein d designates that the sequence has standard DNA conformation and L designates that the sequence is in a locked nucleic acid (LNA) conformation.
20. The reaction mixture of claim 18 , wherein the SBP′ comprises dT 14 .