Highly selective nucleic acid amplification primers

An amplification and detection method that is capable of detecting as few as 10 copies of at least one rare, mutant DNA target sequence in a mixture containing, for each mutant target sequence, 100,000 copies of a closely related wild-type DNA target sequence, comprising

• (a) repeatedly cycling a reaction mixture in a primer-dependent amplification reaction having a primer-annealing temperature, said reaction mixture including said at least one mutant target sequence or its closely related wild-type target sequence or both, a DNA polymerase, other reagents needed for amplification, and for each mutant target sequence a primer pair that includes a multi-part primer comprising, in the 5′ to 3′ direction the following three contiguous DNA sequences:
• an anchor sequence that hybridizes with the mutant target sequence and with its closely related wild-type target sequence during primer annealing;
• a bridge sequence at least six nucleotides long that does not hybridize to either the mutant target sequence or its closely related wild-type target sequence during primer annealing;
• and
• a foot sequence that is at least five nucleotides long, perfectly complementary to the mutant sequence and mismatched to its wild-type sequence by one or two nucleotides, wherein:
• (i) if the anchor sequence and the foot sequence of the primer are hybridized either to the mutant target sequence or to its closely related wild-type target sequence, there is in the target sequence an intervening sequence that does not hybridize to the primer's bridge sequence during primer-annealing, and the bridge sequence and the intervening sequence, together create a bubble in the hybrid having a circumference of 16-52 nucleotides, wherein the intervening sequence is at least six nucleotides long and the bridge sequence has a length equal to or greater than that of the intervening sequence,
• (ii) the circumference of the bubble and the length of the foot sequence in combination result in a weak foot/mutant-target-sequence hybrid that makes copying the mutant target sequence unlikely,
• (iii) the bridge sequence and the foot sequence do not together prime non-target sequences in the mixture, and
• (iv) the probability that during said cycling a multi-part primer/wild-type target sequence hybrid will be extended is at least 10,000 times lower than the probability that during said cycling a multi-part primer/mutant target sequence hybrid will be extended, as evidenced by a ΔCT of at least 13.3 cycles;
• and
• (b) detecting amplified product or products with a dsDNA binding dye, or for each multi-part primer a fluorescent hybridization probe that signals upon hybridization to the amplification product of the primer, or for each multi-part primer a quenched, fluorescently labeled oligonucleotide hairpin at the primer's 5′ end that fluoresces only when incorporated in or hybridized to the primer's amplified product,
• wherein copying is “unlikely” means that, if the reaction were begun with a reaction mixture containing 106 copies of the at least one mutant DNA target sequence, no wild-type sequence and the multi-part primer, and separately with a reaction mixture containing 106 copies of the at least one mutant DNA target sequence, no wild-type sequence and a corresponding conventional primer in place of the multi-part primer, the threshold cycle (CT) of the reaction utilizing the multi-part primer would be delayed by at least three cycles compared to the CT of the reaction utilizing the conventional primer.