Patent ID: 7579154

Claim:
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, and wherein the primer has a general formula of 5′-X p -Y q -Z r -3′, wherein X p represents said 5′-end portion having said pre-selected arbitrary nucleotide sequence substantially not complementary to any site on the template nucleic acid; Y q represents said regulator portion comprising at least three contiguous universal base; Z r 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, and wherein Z r comprises at least 10 contiguous deoxythymidine nucleotides.