Methods of nucleic acid target capture
View Patent ↗Methods for efficiently capturing a target nucleic acid from a sample by using a mixture that contains a capture probe specific for the target nucleic acid, the target nucleic acid, and a denaturant chemical, which mixture is incubated at elevated temperature for a short time, are disclosed. Compositions that include a capture probe that specifically binds to a target nucleic acid and a denaturant chemical, which when mixed with the target nucleic acid and incubated at elevated temperature for a short time, promote efficient hybridization of the capture probe and target nucleic acid are disclosed.
1. A method for isolating a target nucleic acid of interest from a sample, comprising:
mixing a sample containing a target nucleic acid with a capture probe that hybridizes specifically to a target sequence in the target nucleic acid in a detergent-containing solution phase that contains imidazole in a concentration range of 0.5 M to about 4.2 M and an immobilized probe that binds specifically to the capture probe, to provide a reaction mixture,
incubating the reaction mixture at a first temperature in a range of about 85° C. to 95° C. for about 15 minutes or less,
incubating the reaction mixture at a second temperature in a range of about 25° C. to 42° C. for about 20 minutes or less, thereby forming a hybridization complex made up of the capture probe hybridized specifically to the target nucleic acid and the immobilized probe bound specifically to the capture probe, wherein the hybridization complex is attached to a support via the immobilized probe, and
separating the hybridization complex attached to the support from other sample components.
2. The method of claim 1 , wherein the solution phase contains imidazole at a concentration from 0.5 M to 4.2 M, and incubating at the first temperature is for about 1 to 15 minutes.
3. The method of claim 1 , wherein the solution phase contains imidazole at a concentration from 3.0 M to 3.5 M, and incubating at the first temperature is for about 1 to 15 minutes.
4. The method of claim 1 , wherein the solution phase contains imidazole at a concentration from 2.0 M to 2.7 M and incubating at the first temperature is about 90° C. to 95° C. for about 3 to 10 minutes.
5. The method of claim 1 , wherein the solution phase contains imidazole at 2.7 M, incubating at the first temperature is for about 3 to 15 minutes, and the method further includes incubating the reaction mixture at about 60° C. for about 20 minutes between the first and second incubating steps.
6. The method of claim 1 , wherein incubating at the first temperature is about 95° C. for about 3 to 15 minutes.
7. The method of claim 1 , wherein the target nucleic acid is completely or partially double-stranded nucleic acid, or a nucleic acid that includes other secondary or tertiary structure.
8. The method of claim 1 , wherein the capture probe is made up of a target-specific sequence that binds to the target nucleic acid and a tail region that binds to the immobilized probe via a specific binding partner.
9. The method of claim 8 , wherein the tail region binds to the immobilized probe by hybridizing specifically to a complementary sequence of the immobilized probe.
10. The method of claim 1 , further comprising after the separating step, a step of detecting the target nucleic acid or an in vitro amplification product made from the target nucleic acid.
11. A method for isolating a target nucleic acid of interest from a sample, comprising:
mixing a sample containing a target RNA with a nucleic acid capture probe that contains a first sequence that hybridizes specifically to a target sequence in the target RNA in a solution phase that contains a detergent and imidazole in a concentration range of 1.7 M to 2.7 M and an immobilized probe that binds specifically to the capture probe, to provide a reaction mixture,
first incubating the reaction mixture at 85° C. to 95° C. for 3 to 15 minutes,
second incubating the reaction mixture at about 25° C. to 42° C. for about 5 to 15 minutes, thereby forming a hybridization complex made up of the capture probe hybridized specifically to the target RNA and the immobilized probe hybridized specifically to the capture probe, wherein the hybridization complex is attached to a support via the immobilized probe, and
separating the hybridization complex attached to the support from other sample components, thereby isolating the target RNA from other sample components.
12. The method of claim 11 , wherein the second incubating step is at about 25° C. for about 5 minutes.
13. The method of claim 11 , wherein the method further includes incubating the reaction mixture at 60° C. for about 20 minutes between the first and the second incubating step which is at about 25° C. for about 15 minutes.
14. The method of claim 1 , wherein the solution phase contains imidazole at a concentration from 1.7 M to 3.2 M and the incubating step at the first temperature is for about 3 to 7 minutes.
15. The method of claim 1 , wherein the mixing step includes a first capture probe that hybridizes specifically to a first target sequence and a second capture probe that hybridizes specifically to a second target sequence which is different from the first target sequence and wherein the incubating and separating steps are performed by using one set of target capture conditions.
16. A method for isolating a target nucleic acid of interest from a sample, comprising:
mixing a sample containing the target nucleic acid with a nucleic acid capture probe that contains a first sequence that hybridizes specifically to a target sequence in the target nucleic acid in a detergent-containing solution phase that contains about 1.7 M to 2.7 M imidazole and an immobilized probe that binds specifically to the capture probe, to provide a reaction mixture,
incubating the reaction mixture at a first temperature of about 85° C. to 95° C. for about 10 to 15 minutes,
then incubating the reaction mixture at a second temperature of about 60° C. for about 20 minutes,
then incubating the reaction mixture at a third temperature of about 25° C. for about 5 to 15 minutes, thereby forming a hybridization complex made up of the capture probe hybridized specifically to the target nucleic acid and the immobilized probe hybridized specifically to the capture probe, wherein the hybridization complex is attached to a support via the immobilized probe, and
separating the hybridization complex attached to the support from other sample components, thereby isolating the target nucleic acid from other sample components.
17. The method of claim 16 , wherein the solution phase contains imidazole at a concentration of about 1.7 M.
18. The method of claim 16 , wherein the solution phase contains imidazole at a concentration of about 2.0 M.
19. The method of claim 16 , wherein the solution phase contains imidazole at a concentration of about 2.7 M.
20. The method of claim 16 , wherein the mixing step includes a first capture probe that hybridizes specifically to a first target sequence in a first target nucleic acid and a second capture probe that hybridizes specifically to a second target sequence in a second target nucleic acid, thereby permitting formation of two different hybridization complexes under the same incubation conditions, and permitting separation of two different hybridization complexes attached to the support from other sample components, thereby isolating the first target nucleic acid and the second target nucleic acid from other sample components.