Method of nucleic acid sequence detection and nucleic acid sequence detection substrate

A method of nucleic acid sequence detection for detecting the presence of a particular target nucleic acid sequence to be detected in a sample, the method comprising:

• designing first and second oligonucleotide strands in advance such that the second oligonucleotide strand has a complementary nucleic acid sequence to an elongation reaction product dissociated from a duplex formed through elongation reaction using the first oligonucleotide strand as a primer and the target nucleic acid sequence as a template after complementary binding of the target nucleic acid sequence and the first oligonucleotide strand;
• immobilizing a plurality of the designed first and second oligonucleotide strands onto a substrate;
• mixing the sample containing the target nucleic acid sequence with a ligand as a constituent of a chromogenic substance to prepare a mixture solution;
• adding the mixture solution to a reaction system on the substrate and setting the temperature of the reaction system to a heat denaturation temperature at which the target nucleic acid sequence is heat-denatured;
• setting the temperature of the reaction system to a first elongation reaction temperature at which the first oligonucleotide strand complementarily binds to the target nucleic acid sequence for first elongation reaction;
• setting the temperature of the reaction system to a first dissociation temperature at which the first elongation reaction product is dissociated from the first duplex formed through the first elongation reaction;
• setting the temperature of the reaction system to a second elongation reaction temperature at which the dissociated first elongation reaction product complementarily binds to the second oligonucleotide strand for second elongation reaction;
• setting the temperature of the reaction system to a second dissociation temperature at which the second elongation reaction product is dissociated from the second duplex formed through the second elongation reaction;
• setting the temperature of the reaction system to a temperature at which the dissociated second elongation reaction product complementarily binds to the first oligonucleotide strand;
• capturing the ligand within a reticulated space formed on the substrate;
• adding, onto the substrate, an active substance as another constituent of the chromogenic substance and setting the temperature to a temperature for reaction with the ligand;
• and
• detecting a color signal derived from coloring by the chromogenic substance.

A method of detecting a target nucleic acid when present in a sample, comprising:

• contacting the sample and a substrate, the substrate having a plurality of first and second oligonucleotides immobilized on the substrate, the first oligonucleotide comprising a sequence complementary to a sequence of the target nucleic acid and the second oligonucleotide comprising a sequence complementary to a sequence of a first elongation reaction product;
• when the target nucleic acid is present in the sample:
• forming a reticulated space from a plurality of intertwined, cross-linked structures, wherein forming the reticulated space comprises performing the following a plurality of times:
• binding the first oligonucleotide to the target nucleic acid;
• performing an elongation reaction using the first oligonucleotide as a primer and the target nucleic acid as a template to obtain the first elongation reaction product;
• dissociating the first elongation reaction product from the target nucleic acid;
• binding the first elongation reaction product to the second oligonucleotide to form a cross-linked structure between the first and second oligonucleotides;
• performing an elongation reaction using the second oligonucleotide as a primer and the first elongation reaction product as a template to obtain a second elongation reaction product;
• dissociating the second elongation reaction product from the first elongation reaction product;
• binding the second elongation reaction product to a different first oligonucleotide to form a cross-linked structure between the first oligonucleotide and the second oligonucleotide;
• and
• performing an elongation reaction using the first oligonucleotide as a primer and the second elongation reaction product as a template;
• capturing a ligand within the reticulated space;
• and
• producing a detectable signal indicative of the presence of the target nucleic acid in the sample by way of the captured ligand and an active substance.

A method of detecting a target nucleic acid when present in a sample, comprising:

• mixing the sample with a ligand to prepare a mixture;
• applying the mixture to a substrate, the substrate having a plurality of first and second oligonucleotides immobilized on the substrate, the first oligonucleotide comprising a sequence complementary to a sequence of the target nucleic acid and the second oligonucleotide comprising a sequence complementary to a sequence of a first elongation reaction product;
• when the target nucleic acid is present in the sample:
• heat denaturing the target nucleic acid;
• forming a reticulated space from a plurality of intertwined, cross-linked structures, wherein forming the reticulated space comprises performing the following a plurality of times:
• binding the first oligonucleotide to the target nucleic acid;
• performing an elongation reaction using the first oligonucleotide as a primer and the target nucleic acid as a template to obtain the first elongation reaction product;
• dissociating the first elongation reaction product from the target nucleic acid;
• binding the first elongation reaction to the second oligonucleotide to form a cross-linked structure between the first and second oligonucleotides;
• performing an elongation reaction product using the second oligonucleotide as a primer and the first elongation reaction product as a template to obtain a second elongation reaction product;
• dissociating the second elongation reaction product from the first elongation reaction product;
• binding the second elongation reaction product to a different first oligonucleotide to form a cross-linked structure between the first oligonucleotide and the second oligonucleotide;
• and
• performing an elongation reaction using the first oligonucleotide as a primer and the second elongation reaction product as a template;
• capturing the ligand within the reticulated space;
• adding an active substance that binds to or reacts with the ligand;
• and
• producing a detectable signal indicative of the presence of the target nucleic acid in the sample by way of the ligand and the active substance.