# US 10,870,675 B2 · Process using dual specificity oligonucleotide and dual specificity oligonucleotide

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- Number: US 10,870,675 B2
- Document ID: US10870675B2
- Status: Granted
- Kind: B2
- Application number: 15/865,554
- Filed: 2018-01-09
- Published: 2018-05-24
- Granted: 2020-12-22
- Earliest claimed: 2005-03-05
- Assignees: Seegene, Inc.
- Applicants: Seegene, Inc.
- Inventors: Jong Yoon Chun
- Web page: https://patentgrep.com/patent/US10870675B2

## CPC

- C07H 21/04: with deoxyribosyl as saccharide radical
- C07H 21/02: with ribosyl as saccharide radical
- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C12Q 1/6853: using modified primers or templates
- C12Q 1/6858: Allele-specific amplification
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present invention relates to various processes by a template-dependent extension reaction using a dual specificity oligonucleotide and a dual specificity oligonucleotide composed of three different Tm portions therefor. Demonstrated in the present invention are the features of the dual specificity oligonucleotide, which are high hybridization specificity and mismatch tolerance.

## Claims

### Claim 1

A primer set for selectively amplifying a target nucleic acid sequence comprising a pair of dual specificity oligonucleotides represented by the following general formula:
5′-Xp-Yq-Zr-3′ wherein,
Xp represents a 5′-high Tm specificity portion having a hybridizing nucleotide sequence substantially complementary to a site on an initial target nucleic acid to hybridize therewith;
Yq represents a separation portion consisting of contiguous universal bases;
Zr represents a 3′-low Tm specificity portion having a hybridizing nucleotide sequence substantially complementary to a site on the initial target nucleic acid to hybridize therewith;
wherein the initial target template nucleic acid is a nucleic acid molecule present in a sample to be analyzed prior to an amplification reaction using the pair of dual specificity oligonucleotides;
p, q and r represent the number of nucleotides;
p represents an integer of at least 15;
q represents an integer of at least 5;
r represents an integer of at least 3;
X, Y, and Z are deoxyribonucleotides or ribonucleotides;
the Tm of the 5′-high Tm specificity portion is higher than that of the 3′-low Tm specificity portion;
the separation portion has the lowest Tm of the three portions;
the 5′-high Tm specificity portion is longer than the 3′-low Tm specificity portion;
the separation portion forms a non-base-pairing under conditions that the 5′-high Tm specificity portion and the 3′-low Tm specificity portion are annealed to the target nucleic acid, enabling the 5′-high Tm specificity portion to separate from the 3′-low Tm specificity portion in terms of annealing specificity to the target nucleic acid;
whereby the annealing specificity of the dual specificity oligonucleotide is determined dually by the 5′-high Tm specificity portion and the 3′-low Tm specificity portion.

### Claim 2 (depends on 1)

The primer set of claim 1, wherein the universal bases in the separation portion are selected from the group consisting of deoxyinosine, inosine, 7-deaza-2′-deoxyinosine, 2-aza-2′-deoxyinosine, 2′-OMe inosine, 2′-F inosine, deoxy-3-nitropyrrole, 3-nitropyrrole, 2′-OMe 3-nitropyrrole, 2′-F 3-nitropyrrole, 1-(2 ′-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole, deoxy-5-nitroindole, 5-nitroindole, 2′-OMe 5-nitroindole, 2′-F 5-nitroindole, deoxy-4-nitrobenzimidazole, 4-nitrobenzimidazole, deoxy-4-aminobenzimidazole, 4-aminobenzimidazole, deoxynebularine, 2′-F nebularine, 2′-F 4-nitrobenzimidazole, PNA-5-introindole, PNA-nebularine, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole, morpholino-nebularine, morpholino-inosine, morpholino-4-nitrobenzimidazole, morpholino-3-nitropyrrole, phosphoramidate-5-nitroindole, phosphoramidate-nebularine, phosphoramidate-inosine, phosphoramidate-4-nitrobenzimidazole, phosphoramidate-3-nitropyrrole, 2′-O-methoxyethyl inosine, 2′-O-methoxyethyl nebularine, 2′-O-methoxyethyl 5-nitroindole, 2′-O-methoxyethyl 4-nitro-benzimidazole, 2′-O-methoxyethyl 3-nitropyrrole, and combinations thereof.

### Claim 3 (depends on 2)

The primer set of claim 2, wherein the universal bases are deoxyinosine, 1-(2′-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole or 5-nitroindole.

### Claim 4 (depends on 3)

The primer set of claim 3, wherein the universal bases are deoxyinosine.

### Claim 5 (depends on 1)

The primer set of claim 1, wherein the 3′-low Tm specificity portion has the hybridizing nucleotide sequence perfectly complementary to the site on the target nucleic acid to hybridize therewith.

### Claim 6 (depends on 1)

The primer set of claim 1, wherein p represents an integer of 15 to 40.

### Claim 7 (depends on 6)

The primer of claim 6, wherein p represents an integer of 15 to 25.

### Claim 8 (depends on 1)

The primer set of claim 1, wherein q represents an integer of 5 to 10.

### Claim 9 (depends on 1)

The primer set of claim 1, wherein r represents an integer of 3 to 15.

### Claim 10 (depends on 1)

The primer set of claim 1, wherein p is an integer of 15 to 25, q is an integer of 5 to 10, and r is an integer of 3 to 15.

### Claim 11 (depends on 1)

The primer set of claim 1, wherein the Tm of the 5′-high Tm specificity portion ranges from 40° C. to 80° C.

### Claim 12 (depends on 1)

The primer set of claim 1, wherein the Tm of the 3′-low Tm specificity portion ranges from 10° C. to 40° C.

### Claim 13 (depends on 1)

The primer set of claim 1, wherein the Tm of the separation portion ranges from 3° C. to 10° C.