Compositions and methods for capturing target nucleic acids
Capture mixtures and activated capture mixtures are provided that are useful for nucleic acid separation and purification are provided. The mixtures comprise lithium lauryl sulfate, lithium hydroxide, a zwitterionic sulfonic acid buffering agent, and optionally, proteinase K, capture probes comprising a first specific binding partner (SBP), and a second specific binding partner immobilized to a solid support. Related combinations, methods, uses, and kits, are also provided.
1 . A capture mixture for isolating a target nucleic acid from a sample, said capture mixture comprising:
(a) lithium lauryl sulfate;
(b) lithium hydroxide; and
(c) a zwitterionic sulfonic acid buffering agent;
wherein:
(i) the lithium lauryl sulfate is present at a concentration of about 35-120 g/L; and
(ii) the lithium hydroxide is present at a concentration of about 14-50 g/L;
and further wherein:
(iii) the capture mixture comprises a C 2-6 dicarboxylic acid; and/or
(iv) the capture mixture comprises proteinase K; and/or
(v) the capture mixture comprises (1) a first population of capture probes, comprising a first region that is at least about 12 residues in length and comprises at least one poly (k) sequence comprising a randomized sequence comprising G and U/T nucleotides, or a non-randomized repeating (G and U/T) sequence, or a combination thereof; and a second region comprising a first specific binding partner (SBP), wherein the SBP is capable of specifically binding a second specific binding partner (SBP2); and (2) a second population of capture probes, comprising a first region that is at least about 12 residues in length and comprises a poly (r) sequence comprising a randomized sequence comprising G and A nucleotides, a non-randomized repeating (A and G) sequence, or a combination thereof; and a second region comprising a third specific binding partner (SBP3), wherein the SBP3 is capable of specifically binding the SBP2; and/or
(vi) the capture mixture comprises a solid support comprising the SBP2 immobilized thereto; wherein the solid support is beads and the beads are present at a concentration of about 0.15%-1% by weight relative to total solids in the mixture or about 300-2800 mg/L; and/or
(vii) the capture mixture does not comprise one or more of a phosphate buffer, a chelator, or LiCl.
2 . The capture mixture of claim 1 , wherein the capture mixture comprises water.
3 . The capture mixture of claim 1 , wherein the zwitterionic sulfonic acid buffering agent is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) present at a concentration of about 50%-85%, or about 52.5%-57.5%, or about 64%-70%, or about 72%-82% relative to the total solids in the mixture, or a concentration of about 150-275 g/L, or about 175-260 g/L, or about 170-200 g/L, or about 244 g/L, or about 188 g/L.
4 . The capture mixture of claim 2 , further comprising an anti-foaming agent, optionally wherein the anti-foaming agent is a 3-dimensional siloxane defoamer, and the 3-dimensional siloxane defoamer is emulsified in water, wherein the anti-foaming agent is present at a concentration of from about 0.02% w/v to about 0.1% w/v, or from about 0.03% w/v to about 0.08% w/v, or from about 0.03% w/v to about 0.05% w/v, or about 0.05% w/v, or about 0.04% w/v.
5 . The capture mixture of claim 1 , comprising:
(a) lithium lauryl sulfate;
(b) lithium hydroxide;
(c) the zwitterionic sulfonic acid buffering agent;
(d) the C 2-6 dicarboxylic acid; and
(e) proteinase K.
6 . A pre-mix formulation comprising the capture mixture of claim 1 , wherein the pre-mix formulation does not comprise proteinase K, and when the formulation comprises water, the components are present at 1.3X the listed solution concentrations.
7 . The pre-mix formulation of claim 6 , comprising about 244 g/L HEPES, about 39 g/L lithium lauryl sulfate, about 37 g/L lithium hydroxide, about 35 g/L succinic acid, about 0.04% w/v anti-foaming agent, about 3.7 mg/mL each of the first population of capture probes and the second population of capture probes, and about 780 mg/L of SBP2 immobilized on beads.
8 . An activated capture mixture comprising the capture mixture of claim 1 , wherein the capture mixture comprises proteinase K.
9 . An activated capture mixture prepared by combining the pre-mix formulation of claim 6 and proteinase K.
10 . A method for isolating a target nucleic acid from a sample, the method comprising:
a. contacting the capture mixture of claim 1 or the activated capture mixture of claim 8 which includes the first population of capture probes with a solution comprising a target nucleic acid to form a reaction mixture;
b. incubating the reaction mixture in conditions that allow hybridization of the first region of the first population with the target nucleic acid;
c. simultaneously with or subsequently to step (b), incubating the reaction mixture with a solid support comprising the SBP2 immobilized thereto in conditions that allow for association of the SBP and the SBP3 with the SBP2 immobilized to the support, thereby forming a hybridization complex in contact with a solution phase; and
d. separating the support from the solution phase, thereby isolating the target nucleic acid from other components in the sample.
11 . The method of claim 10 , wherein the target nucleic acid is derived from a sample that contains cells and the method comprises treating the cells before the contacting step to release intracellular components into the solution.
12 . The method of claim 11 , wherein treating comprises treating the sample with a detergent.
13 . A kit comprising:
(a) a first component comprising the capture mixture of claim 1 ; and
(b) a second component comprising lithium hydroxide.
14 . A kit comprising:
a first component comprising a pre-mix formulation, wherein (i) the pre-(a) mix formulation comprises the capture mixture of claim 1 , the pre-mix formulation does not comprise proteinase K, and when the formulation comprises water, the components are optionally present at 1.3X the listed solution concentrations, or (ii) the pre-mix formulation is the pre-mix formulation of claim 6 ;
(b) a second component comprising lithium hydroxide; and
(c) a third component comprising proteinase K.