Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer
Demonstration of the advantage of using non-symmetric primer concentrations (as opposed to using symmetric primer concentrations) in multiplex PCR assays that quantitate different mutations that, although occurring in different cells, are located in the same codon.
These assays contained fine-tuned SuperSelective primers BRAF V600E 32-30-10/9-6:1:1 and BRAF V600R 32-30-12/9-5:2:1 (where the underlined value reflects the length of the 5'-tag sequence), and two differently colored molecular beacon probes to detect the resulting amplicons. The left-hand panels show the results from the fluorescein-labeled BRAF V600R-specific molecular beacons (green curves); and the right-hand panels show the results from the Quasar® 670-labeled BRAF V600E-specific molecular beacons (blue curves). Samples without any templates were also run as controls (dotted orange curves). The symmetric PCR reactions, whose results are shown in the upper panels, contained 500 nM of each SuperSelective primer and 1,000 nM of the conventional BRAF common reverse primer. The non-symmetric PCR reactions, whose results are shown in the lower panels, contained only 60 nM of each SuperSelective primer and 1,000 nM of the conventional BRAF common reverse primer.