Annealing control primer and its uses
First Claim
1. A method for improving the annealing specificity of a primer in nucleic acid amplification, comprising the steps of:
- (a) preparing a 3′
-end portion of the primer having a hybridizing nucleotide sequence substantially complementary to a site on a template nucleic acid to hybridize therewith;
(b) forming at the 5′
-end of the 3′
-end portion a regulator portion comprising at least three contiguous universal bases to have the lowest Tm in the three portions of the primer, wherein the regulator portion restricts a primer annealing site to the 3′
-end portion to improve the annealing specificity of the 3′
-end portion; and
(c) forming at the 5′
-end of the regulator portion a 5′
-end portion having a pre-selected arbitrary nucleotide sequence substantially not complementary to any site on the template nucleic acid,whereby the primer has three distinct portions and the annealing specificity of the primer is improved by the presence of the regulator portion and the 5′
-end portion, andwherein the primer has a general formula of 5′
-Xp-Yq-Zr-3′
, wherein Xp represents said 5′
-end portion having said pre-selected arbitrary nucleotide sequence substantially not complementary to any site on the template nucleic acid;
Yq represents said regulator portion comprising at least three contiguous universal base;
Zr represents said 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; and
wherein X, Y and Z is deoxyribonucleotide or ribonucleotide, andwherein Zr comprises at least 10 contiguous deoxythymidine nucleotides.
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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.
9 Citations
19 Claims
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1. A method for improving the annealing specificity of a primer in nucleic acid amplification, comprising the steps of:
-
(a) preparing a 3′
-end portion of the primer having a hybridizing nucleotide sequence substantially complementary to a site on a template nucleic acid to hybridize therewith;(b) forming at the 5′
-end of the 3′
-end portion a regulator portion comprising at least three contiguous universal bases to have the lowest Tm in the three portions of the primer, wherein the regulator portion restricts a primer annealing site to the 3′
-end portion to improve the annealing specificity of the 3′
-end portion; and(c) forming at the 5′
-end of the regulator portion a 5′
-end portion having a pre-selected arbitrary nucleotide sequence substantially not complementary to any site on the template nucleic acid,whereby the primer has three distinct portions and the annealing specificity of the primer is improved by the presence of the regulator portion and the 5′
-end portion, andwherein the primer has a general formula of 5′
-Xp-Yq-Zr-3′
, wherein Xp represents said 5′
-end portion having said pre-selected arbitrary nucleotide sequence substantially not complementary to any site on the template nucleic acid;
Yq represents said regulator portion comprising at least three contiguous universal base;
Zr represents said 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; and
wherein X, Y and Z is deoxyribonucleotide or ribonucleotide, andwherein Zr comprises at least 10 contiguous deoxythymidine nucleotides. - View Dependent Claims (2, 3)
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4. A method for improving the annealing specificity of a first primer in nucleic acid amplification, which comprises the steps of:
-
(a) preparing a 3′
-end portion of the first primer having a hybridizing nucleotide sequence substantially complementary to a site on a template nucleic acid to hybridize therewith at a first annealing temperature in said amplification;(b) forming at the 5′
-end of the 3′
-end portion a regulator portion comprising at least three contiguous universal bases to have the lowest Tm in the three portions of the primer, wherein the regulator portion restricts a primer annealing site to the 3′
-end portion to improve the annealing specificity of the 3′
-end portion at a first annealing temperature in said amplification; and(c) forming at the 5′
-end of the regulator portion a 5′
-end portion having a pre-selected arbitrary nucleotide sequence that serves as a priming site at a second annealing temperature in said amplification and is substantially not complementary to any site on the template nucleic acid,(d) annealing the first primer to a target at the first annealing temperature and extending the first primer to form a first primer extension product; (e) synthesizing a polynucleotide complementary to the first primer extension product; (f) annealing a second primer to the portion of said complementary polynucleotide that is complementary to the pre-selected arbitrary nucleotide sequence at the second annealing temperature, and extending the second primer to form a second primer extension product; whereby the first primer has three distinct portions and the annealing specificity of the first primer is improved by the presence of the regulator portion and the 5′
-end portion, andwherein the first annealing temperature and the second annealing temperature are different. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification