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Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer

Fig 6

Fine-tuned primers and molecular beacons utilized in multiplex PCR assays.

The BRAF SuperSelective primers whose sequences are shown here bind to the opposite strand to which the BRAF V600E SuperSelective primers shown in Fig 3 bind. The target sequence of the BRAF V600R SuperSelective primer mismatches the wild-type target sequence (and the BRAF V600E target sequence) at two adjacent nucleotides, so the BRAF V600R SuperSelective primer has two interrogating nucleotides. The BRAF target sequence is 136 base pairs long; and the EGFR target sequence is 90 base pairs long. The unique 32-nucleotide-long tag sequences at the 5' end of each SuperSelective primer is shown in green; the unique bridge sequence within each SuperSelective primer is shown in blue; and the interrogating nucleotides in the 3' foot sequences are shown in red. The complementary arm sequences of each molecular beacon are shown in black; and their probe sequences are shown in green. The differently colored fluorophores linked to the 5' ends of the molecular beacons are Quasar® 670, fluorescein, and CAL Fluor® Red 610; and the quencher moieties linked to the 3' ends of the molecular beacons are Black Hole Quencher®-1, and Black Hole Quencher®-2.

Fig 6