Double-functional oligonucleotide comprising complementary nucleotide sequence, mis-matched nucleotide sequence, reporter, and quencher, and a methods for nucleic acid amplification and measurement using the same

A method for amplifying a target gene sequence having a length of 50 to 1000 base pairs and measuring a fluorescence signal in real time, the method comprising:

• (a) providing a reaction mixture comprising a nucleic acid sample containing the target gene sequence, a polymerase, nucleotides, and an oligonucleotide;
• (b) subjecting the reaction mixture to a nucleic acid amplification reaction under thermal cycling conditions comprising denaturation, annealing, and extension;
• and
• (c) monitoring the fluorescence signal from the reaction mixture in real time during the nucleic acid amplification reaction,
• wherein the oligonucleotide comprises:
• a primer for amplifying the target gene sequence,
• a complementary sequence complementary to the primer,
• an inosine linker attached at its 3′-end to the 5′-terminus of the primer and at its 5′-end to the 3′-terminus of the complementary sequence,
• at least one mis-matched nucleotide within the complementary sequence to form at least one bubble structure in a double-stranded structure formed between the primer and the complementary sequence, and
• a pair of a reporter dye and a quencher molecule disposed within an oligonucleotide sequence,
• wherein, in a single-stranded state of the oligonucleotide, a distance between the reporter dye and the quencher molecule is at least 15 nucleotides, and in a double-stranded state of the oligonucleotide, the distance is 14 nucleotides or less, and
• wherein the oligonucleotide forms a double-stranded structure at temperatures below 40° C., and is denatured into a single-stranded structure at temperatures of 40° C. to 65° C.