Method for separating biotinylated nucleic acid

Patent metadata

Field	Value
Publication number	US-2017356902-A1
Application number	US-201515537953-A
Country	US
Kind code	A1
Filing date	Dec 17, 2015
Priority date	Dec 22, 2014
Publication date	Dec 14, 2017
Grant date	—

Abstract

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.

First claim

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 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 (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 , comprising a reagent which makes pH of the solution containing free biotin after mixing in 7.8 to 9.5, as a constituent reagent.

Assignees

• Wako Pure Chem Ind Ltd

Inventors

• UKEKAWA Ryo

Classifications

• G01N33/53 Primary

  Immunoassay; Biospecific binding assay; Materials therefor · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C07K17/14

  Peptides being immobilised on, or in, an inorganic carrier · CPC title

• C07K14/375 Primary

  from Basidiomycetes · CPC title

• C12Q1/68

  involving nucleic acids · CPC title