Kits for detecting a mutated target base sequence are provided. For example, the kits can be for detecting a target base sequence (A) containing a nucleotide with a mutated base from a nucleic acid sample. The kits contain a fluorescent-labeled detection probe and a competitive probe combination that is to improve detection by reducing noise. The probe combination is produced using a process that includes, for example, determining the base length and the base sequence of each of the fluorescent-labeled detection probe and the competitive probe. The determining can include experimentally determining the amount to be added to the nucleic acid sample of each of the fluorescent-labeled detection probe and the competitive probe. The kits provide a functional result of a first order derivative curve for the control target reaction sample having a substantial peak (maximum value), but a first order derivative curve for the control non-target reaction sample not having a substantial peak, the functional result improving detection by reducing noise.

Kits for detecting a mutated target base sequence are provided. For example, the kits can be for detecting a target base sequence (A) containing a nucleotide with a mutated base from a nucleic acid sample. The kits contain a fluorescent-labeled detection probe and a competitive probe combination that is to improve detection by reducing noise. The probe combination is produced using a process that includes, for example, determining the base length and the base sequence of each of the fluorescent-labeled detection probe and the competitive probe. The determining can include experimentally determining the amount to be added to the nucleic acid sample of each of the fluorescent-labeled detection probe and the competitive probe. The kits provide a functional result of a first order derivative curve for the control target reaction sample having a substantial peak (maximum value), but a first order derivative curve for the control non-target reaction sample not having a substantial peak, the functional result improving detection by reducing noise.

Herein reported are new tricyclic cytidine compounds, such as 8-diethylamino-tC (8-DEA-tC), that respond to DNA and/or RNA duplex formation with up to a 20-fold increase in fluorescent quantum yield as compared with the free nucleoside, depending on neighboring bases. This turn-on response to duplex formation is by far the greatest of any reported nucleoside analogue that can participate in Watson-Crick base pairing. Measurements of the quantum yield of 8-DEA-tC mispaired with adenosine and, separately, opposite an abasic site show that there is almost no fluorescence increase without the formation of correct Watson-Crick hydrogen bonds. Kinetic isotope effects from the use of deuterated buffer show that the duplex protects 8-DEA-tC against quenching by excited state proton transfer. DFT calculations provide a rationale for the observed photophysical properties that is dependent on duplex integrity and the electronic structure of the analogue.

Herein reported are new tricyclic cytidine compounds, such as 8-diethylamino-tC (8-DEA-tC), that respond to DNA and/or RNA duplex formation with up to a 20-fold increase in fluorescent quantum yield as compared with the free nucleoside, depending on neighboring bases. This turn-on response to duplex formation is by far the greatest of any reported nucleoside analogue that can participate in Watson-Crick base pairing. Measurements of the quantum yield of 8-DEA-tC mispaired with adenosine and, separately, opposite an abasic site show that there is almost no fluorescence increase without the formation of correct Watson-Crick hydrogen bonds. Kinetic isotope effects from the use of deuterated buffer show that the duplex protects 8-DEA-tC against quenching by excited state proton transfer. DFT calculations provide a rationale for the observed photophysical properties that is dependent on duplex integrity and the electronic structure of the analogue.

The present invention addresses the issue of providing a target base sequence detection method, etc., whereby a determination can be readily made regarding whether or not a target base sequence is present in a nucleic acid sample. A fluorescent-labeled detection probe and a competitive probe are added to a nucleic acid sample and caused to hybridize with the nucleic acid in the sample, the fluorescence intensity is measured while changing the temperature of the reaction sample, and first order differentiation is performed on a temperature-fluorescence intensity curve. The fluorescent-labeled detection probe and competitive probe base length, base sequence, and amount to be added to nucleic acid samples are determined such that the first order derivative curve for a control target reaction sample including a target base sequence has a peak but the first order derivative curve for a control non-target reaction sample including a non-target base sequence does not substantially have a peak, when: the fluorescent-labeled detection probe and the competitive probe are added to both the control target nucleic acid sample and the control non-target nucleic acid sample; the fluorescence intensity is measured while the temperature of both the obtained control target reaction sample and the control non-target reaction sample are changed; and first order differentiation is performed on a temperature-fluorescence intensity curve.

The present invention addresses the issue of providing a target base sequence detection method, etc., whereby a determination can be readily made regarding whether or not a target base sequence is present in a nucleic acid sample. A fluorescent-labeled detection probe and a competitive probe are added to a nucleic acid sample and caused to hybridize with the nucleic acid in the sample, the fluorescence intensity is measured while changing the temperature of the reaction sample, and first order differentiation is performed on a temperature-fluorescence intensity curve. The fluorescent-labeled detection probe and competitive probe base length, base sequence, and amount to be added to nucleic acid samples are determined such that the first order derivative curve for a control target reaction sample including a target base sequence has a peak but the first order derivative curve for a control non-target reaction sample including a non-target base sequence does not substantially have a peak, when: the fluorescent-labeled detection probe and the competitive probe are added to both the control target nucleic acid sample and the control non-target nucleic acid sample; the fluorescence intensity is measured while the temperature of both the obtained control target reaction sample and the control non-target reaction sample are changed; and first order differentiation is performed on a temperature-fluorescence intensity curve.

Herein reported are new tricyclic cytidine compounds, such as 8-diethylamino-tC (8-DEA-tC), that respond to DNA and/or RNA duplex formation with up to a 20-fold increase in fluorescent quantum yield as compared with the free nucleoside, depending on neighboring bases. This turn-on response to duplex formation is by far the greatest of any reported nucleoside analogue that can participate in Watson-Crick base pairing. Measurements of the quantum yield of 8-DEA-tC mispaired with adenosine and, separately, opposite an abasic site show that there is almost no fluorescence increase without the formation of correct Watson-Crick hydrogen bonds. Kinetic isotope effects from the use of deuterated buffer show that the duplex protects 8-DEA-tC against quenching by excited state proton transfer. DFT calculations provide a rationale for the observed photophysical properties that is dependent on duplex integrity and the electronic structure of the analogue.

Herein reported are new tricyclic cytidine compounds, such as 8-diethylamino-tC (8-DEA-tC), that respond to DNA and/or RNA duplex formation with up to a 20-fold increase in fluorescent quantum yield as compared with the free nucleoside, depending on neighboring bases. This turn-on response to duplex formation is by far the greatest of any reported nucleoside analogue that can participate in Watson-Crick base pairing. Measurements of the quantum yield of 8-DEA-tC mispaired with adenosine and, separately, opposite an abasic site show that there is almost no fluorescence increase without the formation of correct Watson-Crick hydrogen bonds. Kinetic isotope effects from the use of deuterated buffer show that the duplex protects 8-DEA-tC against quenching by excited state proton transfer. DFT calculations provide a rationale for the observed photophysical properties that is dependent on duplex integrity and the electronic structure of the analogue.

The present invention addresses the issue of providing a target base sequence detection method, etc., whereby a determination can be readily made regarding whether or not a target base sequence is present in a nucleic acid sample. A fluorescent-labeled detection probe and a competitive probe are added to a nucleic acid sample and caused to hybridize with the nucleic acid in the sample, the fluorescence intensity is measured while changing the temperature of the reaction sample, and first order differentiation is performed on a temperature-fluorescence intensity curve. The fluorescent-labeled detection probe and competitive probe base length, base sequence, and amount to be added to nucleic acid samples are determined such that the first order derivative curve for a control target reaction sample including a target base sequence has a peak but the first order derivative curve for a control non-target reaction sample including a non-target base sequence does not substantially have a peak, when: the fluorescent-labeled detection probe and the competitive probe are added to both the control target nucleic acid sample and the control non-target nucleic acid sample; the fluorescence intensity is measured while the temperature of both the obtained control target reaction sample and the control non-target reaction sample are changed; and first order differentiation is performed on a temperature-fluorescence intensity curve.

The present invention addresses the issue of providing a target base sequence detection method, etc., whereby a determination can be readily made regarding whether or not a target base sequence is present in a nucleic acid sample. A fluorescent-labeled detection probe and a competitive probe are added to a nucleic acid sample and caused to hybridize with the nucleic acid in the sample, the fluorescence intensity is measured while changing the temperature of the reaction sample, and first order differentiation is performed on a temperature-fluorescence intensity curve. The fluorescent-labeled detection probe and competitive probe base length, base sequence, and amount to be added to nucleic acid samples are determined such that the first order derivative curve for a control target reaction sample including a target base sequence has a peak but the first order derivative curve for a control non-target reaction sample including a non-target base sequence does not substantially have a peak, when: the fluorescent-labeled detection probe and the competitive probe are added to both the control target nucleic acid sample and the control non-target nucleic acid sample; the fluorescence intensity is measured while the temperature of both the obtained control target reaction sample and the control non-target reaction sample are changed; and first order differentiation is performed on a temperature-fluorescence intensity curve.