Annealing control primer and its uses
First Claim
1. An annealing control primer for improving annealing specificity in nucleic acid amplification, having the formula:
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5′
-Xp—
Yq—
Zr-3′
wherein Xp represents a 5′
-end portion consisting of a pre-selected arbitrary nucleotide sequence substantially not complementary to any site on a template nucleic acid;
wherein Yq represents a regulator portion consisting of 5-15 contiguous universal bases;
wherein Zr represents a 3′
-end portion having a hybridizing nucleotide sequence substantially complementary to a site on the template nucleic acid to hybridize therewith;
wherein p, q and r represent the number of nucleotides;
wherein X, Y and Z is deoxyribonucleotide or ribonucleotide;
wherein the base of the deoxyribonucleotide of X is selected from the group consisting of adenine(A), cytosine(C), guanine(G) and thymine(T);
wherein the regulator portion has the lowest Tm in the three portions of the primer, whereby the regulator portion is capable of regulating an annealing portion of the primer in association with annealing temperatures; and
wherein the 5′
-end portion is not involved in annealing to the template nucleic acid under conditions that the 3′
-end portion anneals to the site on the template nucleic acid such that the 5′
-end portion remains unhybridized with the template nucleic acid.
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Accused Products
Abstract
The present invention relates to an annealing control primer for improving annealing specificity in nucleic acid amplification and its applications to all fields of nucleic acid amplification-involved technology. The present primer comprises (a) a 3′-end portion having a hybridizing nucleotide sequence substantially complementary to a site on a template nucleic acid to hybridize therewith; (b) a 5′-end portion having a pre-selected arbitrary nucleotide sequence; and (c) a regulator portion positioned between said 3′-end portion and said 5′-end portion comprising at least one universal base or non-discriminatory base analog, whereby said regulator portion is capable of regulating an annealing portion of said primer in association with annealing temperature.
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Citations
15 Claims
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1. An annealing control primer for improving annealing specificity in nucleic acid amplification, having the formula:
-
5′
-Xp—
Yq—
Zr-3′wherein Xp represents a 5′
-end portion consisting of a pre-selected arbitrary nucleotide sequence substantially not complementary to any site on a template nucleic acid;wherein Yq represents a regulator portion consisting of 5-15 contiguous universal bases; wherein Zr represents a 3′
-end portion having a hybridizing nucleotide sequence substantially complementary to a site on the template nucleic acid to hybridize therewith;wherein p, q and r represent the number of nucleotides; wherein X, Y and Z is deoxyribonucleotide or ribonucleotide; wherein the base of the deoxyribonucleotide of X is selected from the group consisting of adenine(A), cytosine(C), guanine(G) and thymine(T); wherein the regulator portion has the lowest Tm in the three portions of the primer, whereby the regulator portion is capable of regulating an annealing portion of the primer in association with annealing temperatures; and wherein the 5′
-end portion is not involved in annealing to the template nucleic acid under conditions that the 3′
-end portion anneals to the site on the template nucleic acid such that the 5′
-end portion remains unhybridized with the template nucleic acid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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Specification