Novel method of assaying nucleic acid using labeled nucleotide

Abstract

A novel method is provided to assay at least one nucleic acid. According to this method, a nucleic acid polymerization reaction is conducted in a nucleic acid polymerization reaction system, which contains (A) the at least one nucleic acid as a template, (B) at least one nucleotide monomer labeled with at least one label selected from the group consisting of (a) fluorescent dyes, (b) quenchers and (c) immune related substances with a fluorescent dye or quencher contained therein, and (C) at least one nucleic acid-synthesizing enzyme. The template nucleic acid or a nucleic acid, which has been synthesized using the template nucleic acid as a template, is then assayed from a change or an amount of a change in an optical character of the nucleic acid polymerization system. This method makes it possible to specifically and accurately assay at least one nucleic acid, which is contained in a single system and can be an unknown nucleic acid and/or a known nucleic acid, with excellent sensitivity, in short time and with ease.

1 . A method of assaying at least one nucleic acid, which comprises: conducting a nucleic acid polymerization reaction in a nucleic acid polymerization reaction system comprising (A) said at least one nucleic acid as a template, (B) at least one nucleotide monomer labeled with at least one label selected from the group consisting of (a) fluorescent dyes, (b) quenchers and (c) immune related substances with a fluorescent dye or quencher contained therein, and (C) at least one nucleic acid-synthesizing enzyme; and assaying said template nucleic acid or a nucleic acid, which has been synthesized using said template nucleic acid as a template, from a change or an amount of a change in an optical character of said nucleic acid polymerization system.  
     
     
         2 . A method according to  claim 1 , wherein said label is a combination of at least one donor fluorescent dye and at least one acceptor fluorescent dye.  
     
     
         3 . A method according to  claim 1 , wherein said label is a combination of at least one fluorescent dye and at least one quencher.  
     
     
         4 . A method according to  claim 1 , wherein said nucleic acid polymerization system further comprises (D) at least one nucleotide monomer not labeled with any label.  
     
     
         5 . A method according to  claim 1 , wherein said nucleic acid polymerization system further comprises (E) a nucleic acid primer capable of specifically hybridizing to said template nucleic acid and comprising at least one nucleotide monomer.  
     
     
         6 . A method according to  claim 5 , wherein said nucleic acid primer is labeled with (E′) a label as defined in  claim 1 .  
     
     
         7 . A method according to  claim 1 , wherein said nucleic acid polymerization system further comprises a non-labeled nucleotide.  
     
     
         8 . A method according to  claim 7 , wherein said fluorescence-labeled and/or non-labeled nucleotide comprises guanine (g) and/or said template nucleic acid comprises at least one guanine (g).  
     
     
         9 . A method according to  claim 1 , wherein said nucleic acid polymerization system further comprises a non-labeled nucleotide and a fluorescence-labeled nucleic acid primer without any ingredient (B) as defined in  claim 1 .  
     
     
         10 . A method according to  claim 9 , wherein said non-labeled nucleotide comprises a guanine (g) base.  
     
     
         11 . A method according to any one of claims  1 ,  7  and  9 , wherein said non-labeled nucleotide and/or labeled nucleotide is a triphosphate.  
     
     
         12 . A method of assaying a nucleic acid, which comprises: conducting a nucleic acid polymerization reaction in a nucleic acid polymerization system comprising said nucleic acid as a template, at least one dideoxynucleotide monomer labeled with at least one fluorescent dye and/or at least one quencher, and a nucleic acid-synthesizing enzyme; and assaying said template nucleic acid or a nucleic acid polymer, which has been synthesized using said template nucleic acid as a template, from a change or an amount of a change in fluorescence intensity.  
     
     
         13 . A method according to  claim 12 , wherein said nucleic acid polymerization system is a system further comprising a labeled nucleotide or a non-labeled nucleotide or both of them.  
     
     
         14 . A method according to  claim 12 , wherein said nucleic acid polymerization system further comprises a non-labeled nucleic acid primer.  
     
     
         15 . A method according to any one of claims  1 ,  7 ,  9  and  12 , wherein said nucleic acid polymerization system further comprises (F) a fluorescent dye capable of emitting fluorescence upon binding to a nucleic acid.  
     
     
         16 . A method of assaying a nucleic acid, which comprises: conducting a nucleic acid polymerization reaction in a nucleic acid polymerization system comprising said nucleic acid as a template, a non-labeled dideoxynucleotide monomer, a labeled nucleotide, a non-labeled nucleic acid primer, at least one fluorescent dye cable of emitting fluorescence upon binding to a nucleic acid, and a nucleic acid-synthesizing enzyme; 
 and assaying said template nucleic acid or a nucleic acid polymer, which has been synthesized using said template nucleic acid as a template, from a change or an amount of a change in fluorescence intensity.    
     
     
         17 . A method according to  claim 12  or  16 , wherein said non-labeled dideoxynucleotide, labeled dideoxynucleotide, non-labeled nucleotide and/or labeled nucleotide is a triphosphate.  
     
     
         18 . A method according to any one of claims  1 ,  7 ,  9 ,  12  and  16 , wherein said nucleic acid-synthesizing enzyme is at least one enzyme selected from DNA polymerases, RNA polymerases, reverse transcriptases, and modifications thereof.