Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer
Structure of a SuperSelective primer for the detection of BRAF V600E mutant sequences in the presence of abundant BRAF wild-type sequences, and a demonstration of its use in monoplex real-time PCR assays.
(A) SuperSelective primer BRAF V600E 24-14/14-5:1:1 contains a long 5'-anchor sequence that binds strongly to template strands, a short 3'-foot sequence that includes an interrogating nucleotide that is perfectly complementary to the corresponding nucleotide in a mutant template (but mismatches the corresponding nucleotide in a wild-type template), and a bridge sequence that links the anchor sequence to the foot sequence, and that is chosen to not be complementary to the corresponding intervening sequence in the template strand, thereby forming a single-stranded bubble that separates the function of the anchor from the function of the foot. (B) Real-time PCR assays employing SuperSelective primer BRAF V600E 24-14/14-5:1:1. Six reactions initiated with 106 BRAF wild-type templates plus different quantities of mutant templates (101, 102, 103, 104, 105, and 106) are plotted in blue; a reaction initiated with only 106 wild-type templates is plotted with a dotted orange line; and a control reaction containing no template DNA is plotted in red. (C) The threshold cycle measured for each reaction that contained mutant templates is plotted as a function of the logarithm of the number of mutant templates initially present in each reaction. The dotted orange line indicates the threshold cycle of the reaction containing only wild-type templates.