Assay methods and kits for detecting rare sequence variants

A primer-dependent amplification and detection method that is capable of amplifying and detecting in a sample as few as ten copies of at least one rare DNA intended target sequence (“rare target sequence”) in a mixture containing, for each rare target sequence, 10,000 copies of an unintended target sequence that differs from the rare target sequence by as little as one or two base pairs, comprising:

• (a) preparing a primer-dependent amplification reaction mixture that includes the sample, a DNA polymerase, deoxyribonucleoside triphosphates, an amplification buffer, homogeneous fluorescence detection means for detecting amplification products, and for each rare target sequence a pair of primers consisting of a forward primer and a reverse primer that are specific for the rare target sequence but mismatched to the unintended target sequence,
• (b) repeatedly cycling the primer-dependent amplification reaction mixture by said primer-dependent amplification method to amplify each rare target sequence present in the sample, and
• (c) detecting that rare target sequence by measuring the intensity of fluorescence from the homogeneous fluorescence detection means;
• wherein (i) only two primers are utilized for the detection of each mutation in a rare target sequence and (ii) the forward primer is a SuperSelective primer comprising from the 5′ end to the 3′ end a first anchor sequence, a first bridge sequence, and a first foot sequence that is mismatched to the unintended target sequence by at least its 3′-terminal or 3′-penultimate nucleotide, and (iii) the reverse primer is a SuperSelective primer comprising from the 5′ end to the 3′ end a second anchor sequence, a second bridge sequence, and a second foot sequence that is mismatched to the complement of the unintended target sequence by at least its 3′-terminal or 3′-penultimate nucleotide, and (iv) wherein the pair of primers only initiates synthesis beyond the site where the mutation in the rare target sequence is present.