Patent ID: 8092997

Claim:
A method for synthesizing a nucleic acid molecule using a dual specificity oligonucleotide by a template-dependent extension reaction, comprising: (a) annealing the dual specificity oligonucleotide to a template nucleic acid molecule, wherein the dual specificity oligonucleotide is represented by the formula: 5′-X p -Y q -Z r -3′, wherein, X p is a 5′-high T m specificity portion having a hybridizing nucleotide sequence substantially complementary to a site on the template nucleic acid molecule to hybridize therewith, Y q is a separation portion comprising at least three contiguous universal bases, Z r is a 3′-low T m specificity portion having a hybridizing nucleotide sequence substantially complementary to a site on the template nucleic acid molecule to hybridize therewith, p, q and r are the number of nucleotides, p is an integer of at least 15, q is an integer of at least 3, r is an integer of at least 4, and X, Y, and Z are deoxyribonucleotide or ribonucleotide; wherein the T m of the 5′-high T m specificity portion is at least 20° C. higher than that of the 3′-low T m specificity portion; wherein the separation portion has the lowest T m in the three portions; wherein the 5′-high T m specificity portion is longer than the 3′-low T m specificity portion; wherein the separation portion forms a non base-pairing that does not serve as a hybridizing site and does not interact with the template nucleic acid molecule for base pairing under conditions that the 5′-high T m specificity portion and the 3′-low T m specificity portion are annealed to the template nucleic acid molecule, enabling the 5′-high T m specificity portion to separate from the 3′-low T m specificity portion in terms of annealing specificity to the template nucleic acid molecule, whereby the annealing specificity of the dual specificity oligonucleotide is determined dually by the 5′-high T m specificity portion and the 3′-low T m specificity portion such that the overall annealing specificity of the dual specificity oligonucleotide is enhanced; wherein the annealing is performed under conditions that annealing solely by the 3′-low T m specificity portion does not occur; and wherein 5′-high T m specificity portion and the 3′-low T m specificity portion of the dual specificity oligonucleotide is initially annealed to the template nucleic acid molecule; and (b) extending the dual specificity oligonucleotide to synthesize the nucleic acid molecule complementary to the template nucleic acid.