Method for detecting target nucleic acid and nucleic acid probe used therein

Abstract

A nucleic acid probe for detecting a target nucleic acid. At least one terminal of the probe-binding region in the target nucleic acid is a guanine base, and one or more cytosine bases are present within 1 to 7 bases from the guanine base. The nucleic acid probe comprises an oligonucleotide having a cytosine base facing the guanine base on a terminal and a fluorescent dye conjugated to the cytosine base. The fluorescent dye is quenched by the interaction with a guanine base. The oligonucleotide is completely complementary to the nucleic acid in the probe-binding region except the one or more cytosine bases present within 1 to 7 bases from the terminal guanine base. The base in the oligonucleotide facing the cytosine base closest to the terminal guanine base among the one or more cytosine bases is a base having no fluorescence-quenching effect.

1 . A method for detecting a target nucleic acid, comprising the steps of:
 mixing a nucleic acid probe and a sample to prepare a mixture;   measuring fluorescence intensity from the mixture; and   detecting the target nucleic acid based on the fluorescence intensity,   wherein the target nucleic acid comprises a probe-binding region with which the nucleic acid probe hybridizes, and at least one terminal of the probe-binding region is a guanine, and one or more cytosines are present within 1 to 7 bases from the terminal guanine in the probe-binding region;   wherein the nucleic acid probe comprises an oligonucleotide having a cytosine facing the terminal guanine and a fluorescent dye conjugated to the cytosine, the fluorescent dye being a fluorescent dye that is quenched by interaction with a guanine,   wherein the oligonucleotide is complementary to the nucleic acid in the probe-binding region except for the cytosine that is present within 1 to 7 bases from the terminal guanine and that is closest to the terminal guanine,   wherein the base in the oligonucleotide facing the cytosine closest to the terminal guanine is a base having no fluorescence-quenching effect; and   wherein a decrease in fluorescence intensity of the nucleic acid probe which is not hybridized with the target nucleic acid is reduced compared to the case where the base in the oligonucleotide facing the cytosine closest to the terminal guanine would be a guanine.   
     
     
         2 . The method according to  claim 1 , wherein more than one bases in the oligonucleotide facing the one or more cytosines within the 1 to 7 bases from the terminal guanine have no fluorescence quenching effect. 
     
     
         3 . The method according to  claim 1 , wherein the detection is conducted by melting curve analysis. 
     
     
         4 . The method according to  claim 1 , wherein the sample comprises an amplified product obtained by a nucleic acid amplification reaction using a test nucleic acid as a template. 
     
     
         5 . A method for detecting a target nucleic acid, comprising the steps of:
 mixing a nucleic acid probe and a sample comprising a test nucleic acid to prepare a mixture;   conducting nucleic acid amplification reaction using the test nucleic acid contained in the mixture as a template to obtain an amplified product wherein the amplification reaction is a polymerase chain reaction comprising repeated cycles of a denaturation stage, an annealing stage, and an extension stage;   measuring fluorescence intensity from the mixture in the annealing stage; and   detecting the target nucleic acid based on the fluorescence intensity,   wherein the target nucleic acid comprises a probe-binding region with which the nucleic acid probe hybridizes, and at least one terminal of the probe-binding region is a guanine, and one or more cytosines are present within 1 to 7 bases from the terminal guanine in the probe-binding region;   wherein the nucleic acid probe comprises an oligonucleotide having a cytosine facing the terminal guanine and a fluorescent dye conjugated to the cytosine, the fluorescent dye being a fluorescent dye that is quenched by the interaction with a guanine,   wherein the oligonucleotide is complementary to the nucleic acid in the probe-binding region except for the cytosine that is present within 1 to 7 bases from the terminal guanine, and that is closest to the terminal guanine   wherein the base in the oligonucleotide facing the cytosine closest to the terminal guanine is a base having no fluorescence-quenching effect; and   wherein a decrease in fluorescence intensity of the nucleic acid probe which is not hybridized with the target nucleic is reduced acid compared to the case where the base in the oligonucleotide facing the cytosine closest to the terminal guanine would be a guanine.   
     
     
         6 . A method for detecting a target nucleic acid, comprising the steps of:
 mixing a nucleic acid probe and a sample to prepare a mixture;   measuring fluorescence intensity from the mixture; and   detecting the target nucleic acid based on the fluorescence intensity,   wherein the target nucleic acid comprises a probe-binding region with which the nucleic acid probe hybridizes, and at least one terminal of the probe-binding region is a guanine, and one or more single nucleotide polymorphisms are present within 1 to 7 bases from the terminal guanine in the probe-binding region;   wherein the nucleic acid probe comprises an oligonucleotide having a cytosine facing the terminal guanine and a fluorescent dye conjugated to the cytosine, the fluorescent dye being a fluorescent dye that is quenched by the interaction with a guanine,   wherein the oligonucleotide is complementary to the nucleic acid in the probe-binding region except for the one or more single nucleotide polymorphisms that are present within 1 to 7 bases from the terminal guanine;   wherein one or more bases in the oligonucleotide facing the one or more single nucleotide polymorphisms are a guanine or a base having no fluorescence-quenching effect;   wherein the base in the oligonucleotide facing the single nucleotide polymorphism that is closest to the terminal guanine is a base having no fluorescence-quenching effect; and   wherein a decrease in fluorescence intensity of the nucleic acid probe which is not hybridized with the target nucleic acid is reduced compared to the case where the base in the oligonucleotide facing the single nucleotide polymorphism closest to the terminal guanine would be a guanine.   
     
     
         7 . The method according to  claim 6 , wherein the detection is carried out by melting curve analysis. 
     
     
         8 . The method according to  claim 7 , wherein the single nucleotide polymorphism is detected by melting curve analysis. 
     
     
         9 . The method according to  claim 6 , wherein the sample comprises an amplified product obtained by a nucleic acid amplification reaction using a test nucleic acid as a template.