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
The present disclosure relates to a complementary double-stranded oligo, in which, for the amplification of a particular gene sequence, an inosine linker is linked to the 5′-terminus of a primer for the corresponding sequence, a sequence complementary to the primer is linked to the inosine linker, and at least one mis-matched nucleotide is included in the complementary sequence to form a bubble structure; in which, depending on the treatment temperature, at a predetermined temperature or lower, a single stranded oligo is turned into a double-stranded form to exist in an inactivation form, and at a predetermined temperature or higher, the oligo is activated into a single-stranded oligo; and in which, a fluorescent substance (reporter dye) and a quenching material (quencher molecule) are attached to the oligo, so that the oligo can be applied as a primer or a probe, and thus only two oligos can realize the gene amplification and fluorescent signal real-time measurement with high specificity, and to a measuring method after a fluorescent arrangement step using the oligomer is added. The present disclosure has advantages in view of an oligo and a design method therefor, in which an oligo capable of simultaneously performing amplification and detection with an amplification size of 500 bp to 1000 bp for a particular gene is used to allow a reporter dye and a quencher molecule to be attached to only two target gene amplification sites, thereby confirming whether real-time amplification occurs.
1 . 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.
2 . The method according to claim 1 , wherein the complementary sequence has two or more mis-matched nucleotide.
3 . The method according to claim 1 , wherein, for the pair of the reporter dyes and the quencher molecule in the single stranded structure, and a distance between the reporter dye and the quencher molecule is 15 mer or more.
4 . The method according to claim 1 , wherein the inosine linker is comprised of from 1 to 9 inosine nucleotides.
5 . The method according to claim 1 , wherein the oligonucleotide is used in a forward or reverse location for gene amplification and a real-time PCR.
6 . The method according to claim 1 , wherein the oligonucleotide is one of SEQ ID NOs: 3, 7 to 13, SEQ ID NO: 22, SEQ ID NO: 26, or SEQ ID NO: 33.
7 . The method according to claim 1 , wherein the oligonucleotide comprises one to four bubble structures.