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 and a quenching material 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.
1 . A complementary double-stranded oligonucleotide, wherein, 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 at the 5′-terminus of the linker, and at least one mis-matched nucleotide is included in the complementary sequence site to form a bubble structure; and wherein the oligonucleotide has a double-stranded structure including a pair of a reporter dye and a quencher molecule for detecting nucleic acid for specific gene amplification and is denatured into a single strand at a certain temperature or higher.
2 . The oligonucleotide according to claim 1 , wherein the primer site of the corresponding sequence of a specific gene and the complementary sequence thereof are linked to the inosine linker in one strand of the oligonucleotide, and the complementary sequence has two or more mis-matched nucleotide sequences.
3 . The oligonucleotide according to claim 1 , wherein the melting temperature separated from the double strand to single strand is from 40 to 65° C.
4 . The oligonucleotide according to claim 1 , wherein the oligonucleotide has one to four bubble structures.
5 . The oligonucleotide according to claim 1 , wherein the oligonucleotide has a pair of reporter dyes and quencher molecules in a single strand structure, and the distance between the two is 15mer or more.
6 . The oligonucleotide according to claim 1 , wherein the oligonucleotide is located within 14 mer of the reporter dye and the quencher molecule when the double strand is formed.
7 . The oligonucleotide according to claim 5 , wherein for the oligonucleotide, the location of reporter dye is in the mis-matching sequence and the sequence complementary to the 5′-terminus or in the corresponding sequence of the specific gene from the second of the 3′-terminus.
8 . The oligonucleotide according to claim 5 , wherein for the oligonucleotide, the location of quencher molecule is in the mis-matching sequence and the sequence complementary to the 5′-terminus or in the corresponding sequence of the specific gene from the second of the 3′-terminus.
9 . The oligonucleotide according to claim 1 , wherein the inosine linker is comprised of from 0 to 9 inosine nucleotides.
10 . The oligonucleotide according to claim 1 , wherein the oligonucleotide is used in a forward or reverse location for gene amplification and a real-time PCR, respectively, or together.
11 . The oligonucleotide according to claim 1 , wherein the oligonucleotide is one of SEQ ID NOs.: 2 to 13, SEQ ID NO.: 22, SEQ ID NO.: 26, SEQ ID NO.: 29, or SEQ ID NO.: 33.
12 . A method for gene amplifying a specific gene site from 50 bp to 1000 bp using the oligonucleotide of claim 1 and measuring a fluorescence signal in real time.
13 . (canceled)
14 . (canceled)
15 . (canceled)
16 . (canceled)
17 . A composition for amplification of nucleic acid and measurement of fluorescence amount, the composition comprising the oligonucleotide of claim 1 as an active ingredient.
18 . The composition according to claim 17 , wherein the composition further includes at least one selected from the group consisting of a DNA polymerase having 5′ 3′ exonuclease (+) or (−), or 3′→5′ exonuclease (−) activity, and a Hotstart DNA polymerase.
19 . (canceled)