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

Fig 8

Demonstration of the value of including primers for a reference wild-type gene in non-symmetric multiplex PCR assays employing fine-tuned SuperSelective primers.

The four left-hand panels show the results obtained for reactions containing 10,000 EGFR wild-type fragments, 10,000 BRAF wild-type fragments, 1,000 BRAF V600R fragments, and different quantities of BRAF V600E fragments (0; 10, 39; 156; 625; and 2,500 copies). The four right-hand panels show the results obtained for reactions containing 10,000 EGFR wild-type fragments, 10,000 BRAF wild-type fragments, 1,000 BRAF V600E fragments, and different quantities of BRAF V600R fragments (0; 10, 39; 156; 625; and 2,500 copies). The panels in the top row show the results recorded by the CAL Fluor® Red 610 EGFR wild-type-specific molecular beacon; the panels in the second row show the results recorded by the fluorescein-labeled BRAF V600R-specific molecular beacon; and the panels in the third row show the results recorded by the Quasar® 670-labeled BRAF V600E-specific molecular beacon. The lower panels plot the logarithm of the number of variable BRAF target fragments present in each reaction against the Ct value that was obtained. The Ct values for the 1,000 fragments of the non-variable BRAF mutant in each sample, as well as the Ct values for the 10,000 EGFR reference wild-type fragments present in each sample, are also plotted on the same line. The dotted orange lines indicate the Ct value of the sample containing no BRAF V600E mutant targets (left-hand panel) and no BRAF V600R targets (right-hand panel).

Fig 8

doi: https://doi.org/10.1371/journal.pone.0156546.g008