Method for efficiently detecting double-stranded nucleic acid

Abstract

This invention relates to a method for efficiently detecting double-stranded nucleic acids. More particularly, this invention relates to a method for reducing signals derived from an intercalator bound to a single-stranded nucleic acid, wherein a compound that reacts more preferentially with an intercalator bound to a single-stranded nucleic acid than with an intercalator bound to a double-stranded nucleic acid or a compound that is bound to a single-stranded nucleic acid more strongly than an intercalator and is bound to a double-stranded nucleic acid more weakly than an intercalator is added to a mixture comprising double-stranded and single-stranded nucleic acids both having intercalators bound thereto, thereby reducing signals derived from an intercalator bound to a single-stranded nucleic acid.

1 - 15 . (canceled) 
     
     
         16 . A kit for detecting a product of nucleic acid amplification, the kit comprising:
 an intercalator; and   a compound that reacts more preferentially with an intercalator bound to a single-stranded nucleic acid than with an intercalator bound to a double-stranded nucleic acid or a compound that binds to a single-stranded nucleic acid more strongly than an intercalator and that binds to a double-stranded nucleic acid more weakly than an intercalator.   
     
     
         17 . The kit according to  claim 16 , wherein the intercalator is any of ethidium bromide, acridine orange, TO-PRO-1® (Quinolinium, 4-[(3-methyl-2(3H)-benzothiazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-, diiodide), or YO-PRO-1® (Quinolinium,
 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-, diiodide).   
     
     
         18 . The kit according to  claim 17 , wherein the compound that reacts more preferentially with an intercalator bound to a single-stranded nucleic acid than with an intercalator bound to a double-stranded nucleic acid is an oxidant or a reducer. 
     
     
         19 . The kit according to  claim 18 , wherein the oxidant is any of sodium hypochlorite, hydrogen peroxide, or potassium permanganate. 
     
     
         20 . The kit according to  claim 18 , wherein the reducer is sodium borohydride or sodium cyanoborohydride. 
     
     
         21 . The kit according to  claim 16 , wherein the compound that is bound to a single-stranded nucleic acid more strongly than an intercalator and is bound to a double-stranded nucleic acid more weakly than an intercalator is a second intercalator different from said intercalator. 
     
     
         22 . The kit according to  claim 21 , wherein the second intercalator is any of methylene blue, actinomycin D, SYBR® Green 2 (CAS Registry No. 172827-25-7), or OliGreen® (CAS Registry No. 268220-33-3). 
     
     
         23 . The kit according to  claim 16 , wherein the kit comprises:
 a compound that reacts more preferentially with an intercalator bound to a single-stranded nucleic acid than with an intercalator bound to a double-stranded nucleic acid; and   a compound that binds to a single-stranded nucleic acid more strongly than an intercalator and that binds to a double-stranded nucleic acid more weakly than an intercalator.   
     
     
         24 . The kit according to  claim 16 , further comprising a polymerase. 
     
     
         25 . The kit according to  claim 24 , wherein the polymerase catalyzes strand displacement-type synthesis of complementary chains. 
     
     
         26 . The kit according to  claim 25 , wherein the polymerase is selected from group consisting of Bst DNA polymerase, Bca(exo-) DNA polymerase, the Klenow fragment of  E. coli  DNA polymerase I, Vent DNA polymerase, Vent(Exo-) DNA polymerase, DeepVent DNA polymerase, DeepVent(Exo-) DNA polymerase, φ29 phage DNA polymerase, MS-2 phage DNA polymerase, Z-Taq DNA polymerase, and KOD DNA polymerase. 
     
     
         27 . The kit according to  claim 16 , further comprising a melting temperature regulator. 
     
     
         28 . The kit according to  claim 27 , wherein the melting temperature regulator is betaine, trimethylamine N-oxide, dimethyl sulfoxide, or formamide. 
     
     
         29 . The kit according to  claim 16 , further comprising a primer. 
     
     
         30 . The kit according to  claim 29 , wherein the primer contains (i) a polynucleotide sequence F2 that is complementary to F2c, (ii) a polynucleotide sequence F3 that is complementary to F3c, (iii) a polynucleotide sequence of R2, (iv) a polynucleotide sequence R1c that is complementary to R1; or (v) a polynucleotide sequence of R3,
 wherein F1c, F2c, and F3c are polynucleotide sequences selected in this order from the 3′ end of a target region to be amplified in a polynucleotide chain to the 3′ end of the polynucleotide chain and wherein R1, R2, and R3 are polynucleotide sequences selected in this order from the 5′ end of the target region to the 5′ end of the polynucleotide chain.   
     
     
         31 . The kit of  claim 16 , wherein the product of nucleic acid amplification is double-stranded nucleic acid.