Method for separating biotinylated nucleic acid
An object of the invention is to obtain a biotinylated nucleic acid efficiently, by enhancing dissociation efficiency of biotin in the biotinylated nucleic acid and tamavidin 2 in a tamavidin 2-immobilized insoluble carrier. The inventive method for separating a biotinylated nucleic acid includes (1) contacting a sample containing a biotinylated nucleic acid wherein the biotin is bound to the nucleic acid with a insoluble carrier on which tamavidin is immobilized (a tamavidin-immobilized insoluble carrier) to form a complex of the biotinylated nucleic acid and the tamavidin-immobilized insoluble carrier, and (2) separating the biotinylated nucleic acid from the complex in a solution having pH of 7.8 to 9.5 and in the presence of free biotin. The invention also provides a method for separating the biotinylated nucleic acid to which the nucleic acid-binding protein is bound, a method for separating the nucleic acid-binding protein, and a kit for separating the nucleic acid.
1. A method for separating a biotinylated nucleic acid, comprising the following steps:
(1) a step for contacting a sample containing a biotinylated nucleic acid wherein the biotin is bound to the nucleic acid with an insoluble carrier on which tamavidin is immobilized (a tamavidin-immobilized insoluble carrier) to form a complex of the biotinylated nucleic acid and the tamavidin-immobilized insoluble carrier (step A-1),
(2) a step for separating the biotinylated nucleic acid from the complex obtained in the step A-1, in a solution having pH of 7.8 to 9.5, and in the presence of free biotin (step A-2).
2. The method according to claim 1 , wherein the tamavidin is tamavidin 2-REV.
3. The method according to claim 1 , wherein the step A-1 is carried out in the presence of 40 to 4000 times mol of free biotin relative to the tamavidin.
4. A method for separating a biotinylated nucleic acid to which a nucleic acid-binding protein is bound, comprising the following steps:
(1) a step for contacting a sample containing a protein capable of binding to a nucleic acid (a nucleic acid-binding protein), a biotinylated nucleic acid wherein the biotin is bound to the nucleic acid, and a tamavidin-immobilized insoluble carrier to form a complex of the nucleic acid-binding protein, the biotinylated nucleic acid, and the tamavidin-immobilized insoluble carrier (step B-1),
(2) a step for separating the biotinylated nucleic acid to which the nucleic acid-binding protein is bound from the complex obtained in the step B-1, in a solution having pH of 7.8 to 9.5, and in the presence of free biotin (step B-2).
5. The method according to claim 4 , wherein the tamavidin is tamavidin 2-REV.
6. The method according to claim 4 , wherein the step B-1 is carried out in the presence of 40 to 4000 times mol of free biotin relative to the tamavidin.
7. A method for separating a nucleic acid-binding protein, comprising the following steps:
(1) a step for contacting a sample containing a nucleic acid-binding protein, a biotinylated nucleic acid wherein the biotin is bound to the nucleic acid, and a tamavidin-immobilized insoluble carrier to form a complex of the nucleic acid-binding protein, the biotinylated nucleic acid, and the tamavidin-immobilized insoluble carrier (step C-1),
(2) a step for separating the biotinylated nucleic acid to which the nucleic acid-binding protein is bound from the complex obtained in the step C-1, in a solution having pH of 7.8 to 9.5, and in the presence of free biotin (step C-2),
(3) a step for separating the nucleic acid-binding protein from the biotinylated nucleic acid to which the nucleic acid-binding protein is bound obtained in the step C-2 (step C-3).
8. The method according to claim 7 , wherein the tamavidin is a tamavidin 2-REV.
9. The method according to claim 7 , wherein the step C-1 is carried out in the presence of 40 to 4000 times mol of free biotin relative to the tamavidin.
10. A kit for separating a nucleic acid, comprising a reagent containing an insoluble carrier on which tamavidin is immobilized, and a reagent which makes pH of the solution after mixing in 7.8 to 9.5, as constituent reagents.
11. The kit according to claim 10 , wherein the tamavidin is tamavidin 2-REV.
12. The kit according to claim 10 , wherein the reagent which makes pH of the solution after mixing in 7.8 to 9.5 is a reagent that contains free biotin.
13. The kit according to claim 10 , further comprising a reagent containing free biotin, as a constituent reagent.