Patent ID: 8323895

Claim:
A method for selectively amplifying a target nucleic acid sequence from a DNA or a mixture of nucleic acids, which comprises amplifying the target nucleic acid sequence by performing at least two cycles of primer annealing, primer extending and denaturing using a primer set comprising a pair of dual specificity oligonucleotides with enhanced annealing specificity; wherein the dual specificity oligonucleotide is represented by the following general formula: 5′-X p -Y q -Z r -3′ wherein, Xp represents a 5′-high T m specificity portion having a hybridizing nucleotide sequence substantially complementary to a site on the target nucleic acid sequence to hybridize therewith; Yq represents a separation portion comprising at least three contiguous universal bases; Zr represents a 3′-low T m specificity portion having a hybridizing nucleotide sequence substantially complementary to a site on the target nucleic acid sequence to hybridize therewith; p, q and r represent the number of nucleotides; p represents an integer of at least 15; q represents an integer of at least 3; r represents an integer of at least 3; X, Y, and Z are deoxyribonucleotide or ribonucleotide; the T m of the 5′-high T m specificity portion is higher than that of the 3′-low T m specificity portion; the separation portion has the lowest T m in the three portions; the 5′-high T m specificity portion is longer than the 3′-low T m specificity portion; the separation portion forms a non base-pairing that does not serve as a hybridizing site and not interact with the target nucleic acid sequence 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 target nucleic acid sequence, 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 target nucleic acid sequence; 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 annealing in the amplification reaction is performed under conditions that annealing solely by the 3′-low T m specificity portion does not occur; wherein a first cycle and a second cycle of the amplification reaction involve annealing of both the 5′-high T m specificity portion and the 3′-low T m specificity portion of the dual specificity oligonucleotide to the target nucleic acid sequence.