Methods of nucleic acid target capture

Abstract

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 solution phase that contains a denaturant chemical 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 60° 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 denaturant chemical is 8 M urea and incubating at the first temperature is about 95° C. for about 10 minutes or less. 
     
     
         3 . The method of  claim 1 , wherein the denaturant chemical is imidazole at a concentration from 0.5 M to 4.2 M, and incubating at the first temperature is about 60° C. for about 1 to 15 minutes. 
     
     
         4 . The method of  claim 1 , wherein the denaturant chemical is imidazole at a concentration from 3.0 M to 3.5 M, and incubating at the first temperature is at 60° C. for about 1 to 15 minutes. 
     
     
         5 . The method of  claim 1 , wherein the denaturant chemical is 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. 
     
     
         6 . The method of  claim 1 , wherein the denaturant chemical is imidazole at 2.7 M, incubating at the first temperature is about 75° C. to 95° C. 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. 
     
     
         7 . The method of  claim 6 , wherein incubating at the first temperature is about 95° C. for about 3 to 15 minutes. 
     
     
         8 . 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. 
     
     
         9 . 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. 
     
     
         10 . The method of  claim 9 , wherein the tail region binds to the immobilized probe by hybridizing specifically to a complementary sequence of the immobilized probe. 
     
     
         11 . The method of  claim 1 , further comprising a step of detecting the target nucleic acid or an in vitro amplification product made from the target nucleic acid after separating the hybridization complex attached to the support from other sample components. 
     
     
         12 . 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 solution phase that contains a denaturant chemical and an immobilized probe that binds specifically to the capture probe, to provide a reaction mixture,   incubating the reaction mixture at about 25° C. for about 1 minute to 60 minutes, 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.   
     
     
         13 . The method of  claim 12 , wherein the denaturant chemical is urea at a concentration of about 1 M. 
     
     
         14 . The method of  claim 12 , wherein the denaturant chemical is imidazole at a concentration between 0.05 M and 0.5 M and the incubating step is for about 2 to 30 minutes. 
     
     
         15 . The method of  claim 12 , wherein the denaturant chemical is imidazole at a concentration of about 0.5 M and the incubating step is for about 15 minutes. 
     
     
         16 . A composition for specific capture of a target nucleic acid, comprising at least one target nucleic acid, at least one capture probe that hybridizes specifically to a target sequence in the target nucleic acid, an immobilized probe that binds specifically to the capture probe, and a solution phase hybridization mixture that contains imidazole at a concentration from 0.05 M to 4.2 M or urea at a concentration from 1 to 8 M. 
     
     
         17 . The composition of  claim 16 , wherein the solution phase hybridization mixture contains imidazole at a concentration of from 0.05 to 0.5 M. 
     
     
         18 . The composition of  claim 16 , wherein the solution phase hybridization mixture contains imidazole at a concentration from 1.7 M to 3.5 M. 
     
     
         19 . The composition of  claim 16 , wherein the solution phase hybridization mixture contains imidazole at a concentration from 2.0 M to 2.7 M. 
     
     
         20 . The composition of  claim 16 , wherein the composition 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.