Processes Using Dual Specificity Oligonucleotide and Dual Specificity Oligonucleotide

A method for amplifying two or more target nucleotide sequences simultaneously using two or more pairs of primers in the same reaction, which comprises amplifying the target nucleotide sequences by performing at least two cycles of primer annealing, primer extending and denaturing, using two or more primer pairs in which at least one primer of the primer pair is a dual specificity oligonucleotide, characterized in that the dual specificity oligonucleotides have three portions in which a 5′-high Tm specificity portion has a hybridizing nucleotide sequence substantially complementary to a site on the target nucleotide sequence to hybridize therewith, a separation portion comprises at least two universal bases and a 3′-low Tm specificity portion has a hybridizing nucleotide sequence substantially complementary to a site on the target nucleotide sequence to hybridize therewith, 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 in the three portions;

• the separation portion forms a non base-pairing bubble structure under conditions that the 5′-high Tm specificity portion and the 3′-low Tm specificity portion are annealed to the target nucleotide sequence;
• wherein annealing in the amplification reaction is performed under conditions that annealing solely by the 3′-low Tm specificity portion does not occur.

A method for sequencing a target nucleic acid molecule using a dual specificity oligonucleotide from a DNA or a mixture of nucleic acids, which comprises the steps of:

• (a) synthesizing a complementary nucleic acid molecule to the nucleic acid molecule to be sequenced by performing at least two cycles of primer annealing, primer extending and denaturing, using the dual specificity oligonucleotide as a sequencing primer;
• wherein the dual specificity oligonucleotide has three portions in which a 5′-high Tm specificity portion has a hybridizing nucleotide sequence substantially complementary to a site on the target nucleic acid molecule to hybridize therewith, a separation portion comprises at least two universal bases and a 3′-low Tm specificity portion has a hybridizing nucleotide sequence substantially complementary to a site on the target nucleic acid molecule to hybridize therewith, 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, in the three portions;
• the separation portion forms a non base-pairing bubble structure under conditions that the 5′-high Tm specificity portion and the 3′-low Tm specificity portion are annealed to the target nucleic acid molecule;
• wherein annealing in the synthesizing reaction is performed under conditions that annealing solely by the 3′-low Tm specificity portion does not occur;
• and
• (b) determining a nucleotide sequence of the synthesized complementary nucleic acid molecule.

A method for detecting a nucleic acid molecule with genetic diversity by a template-dependent extension reaction of a dual specificity oligonucleotide, which comprises the steps of:

• (a) annealing a dual specificity oligonucleotide to a template nucleic acid molecule, wherein the dual specificity oligonucleotide has three portions in which a 5-high Tm specificity portion has a hybridizing nucleotide sequence substantially complementary to a site on the template nucleic acid to hybridize therewith, a separation portion comprises at least two universal bases and a 3′-low Tm specificity portion has a hybridizing nucleotide sequence substantially complementary to a site on the template nucleic acid to hybridize therewith, 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 in the three portions;
• the separation portion forms a non base-pairing bubble structure under conditions that the 5′-high Tm specificity portion and the 3′-low Tm specificity portion are annealed to the template nucleic acid, wherein the annealing is performed under conditions that annealing solely by the 3′-low Tm specificity portion does not occur and annealing occurs when the 5′-high Tm specificity portion and/or the 3′-low Tm specificity portion has one or more mismatched bases to its target site;
• (b) extending the dual specificity oligonucleotide to synthesize the nucleic acid molecule complementary to the template;
• and
• (c) detecting the occurrence of the template-dependent extension of the dual specificity oligonucleotide.

A method for detecting a target nucleotide sequence in a nucleic acid sample by a template-dependent extension reaction of a dual specificity oligonucleotide, which comprises the steps of:

• (a) extending a dual specificity oligonucleotide as a probe immobilized on a substrate comprising at least one cycle of a hybridization, a template-dependent extension and a denaturation, wherein the hybridization is performed by contacting the dual specificity oligonucleotide to the nucleic acid sample, wherein the dual specificity oligonucleotide has three portions in which a 5-high Tm specificity portion has a hybridizing nucleotide sequence substantially complementary to a site on the target nucleotide sequence to hybridize therewith, a separation portion comprises at least two universal bases and a 3-low Tm specificity portion has a hybridizing nucleotide sequence substantially complementary to a site on the target nucleotide sequence to hybridize therewith, 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 in the three portions;
• the separation portion forms a non base-pairing bubble structure under conditions that the 5′-high Tm specificity portion and the 3′-low Tm specificity portion are annealed to the target nucleotide sequence, wherein the hybridization is performed under conditions that hybridization solely by the 3′-low Tm specificity portion does not occur;
• and
• (b) analyzing the occurrence of the template-dependent extension.

A dual specificity oligonucleotide for synthesizing a nucleic acid molecule by a template-dependent extension reaction, which is 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 a template nucleic acid to hybridize therewith, Yq represents a separation portion comprising at least two universal bases, Zr represents a 3′-low Tm specificity portion having a hybridizing nucleotide sequence substantially complementary to a site on the template nucleic acid to hybridize therewith, p, q and r represent the number of nucleotides, and X, Y, and Z are deoxyribonucleotide or ribonucleotide;
• 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 in the three portions;
• the separation portion forms a non base-pairing bubble structure under conditions that the 5′-high Tm specificity portion and the 3′-low Tm, specificity portion are annealed to the template 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 template nucleic acid, whereby the annealing specificity of the oligonucleotide is determined dually by the 5′-high Tm specificity portion and the 3′-low Tm specificity portion such that the overall annealing specificity of the oligonucleotide is enhanced.

A method for enabling an annealing specificity of an oligonucleotide to be dually determined through a structure of the oligonucleotide, which comprises the steps of:

• (a) selecting a target nucleic acid sequence;
• (b) designing a sequence of an oligonucleotide having (i) a hybridizing sequence substantially complementary to the target nucleic acid and (ii) a separation portion comprising at least two universal bases, such that the separation portion intervenes in the hybridizing sequence to form three portions in the oligonucleotide;
• and
• (c) determining the position of the separation portion in the oligonucleotide to allow a portion at the 5′-direction of the separation portion to have a higher Tm than a portion at the 3-direction of the separation portion and to allow the separation portion to have the lowest Tm in the three portions, thereby providing an oligonucleotide having three distinct portions with different Tm values from one another in which (i) a 5-high Tm specificity portion of the oligonucleotide has a hybridizing nucleotide sequence substantially complementary to the target nucleic acid, (ii) a 3′-low Tm specificity portion of the oligonucleotide has a hybridizing nucleotide sequence substantially complementary to the target nucleic acid;
• and (iii) the separation portion of the oligonucleotide between the 5′-high Tm specificity portion and 3′-low Tm specificity portion comprises at least two universal bases;
• and the Tm of the 5′-high Tm specificity portion is higher than that of the 3-low Tm specificity portion and the separation portion has the lowest Tm in the three portions,
• whereby the annealing specificity of the oligonucleotide to the target nucleic acid is determined dually by both the 5′-high Tm specificity portion and the 3′-low Tm specificity portion.