Allele-specific PCR for BRAF V600E Detection

Allele-specific PCR was a modified version of the allele-specific locked nucleic acid PCR published by Morandi et al.^{41 (link)}. Briefly, each qPCR reaction included 10 µl of DNA eluate or 10 ng of genomic DNA as positive control (Cat Num. HD238, Horizon Discovery), 1X SsoAdvanced Universal Probes Mastermix (Cat Num. 172-5285, Bio-Rad, US), 0.625 µl of primers (10 µM) and 0.3125 µl of fluorescent probe (10 µM) in a total volume of 25 µl. After careful mixing, each reaction was loaded in triplicate on a 96-well PCR plate and the following qPCR program was launched: 95 °C for 3′, 40 cycles at 95 °C for 5″ and 60 °C for 30″. To calculate the % of recovered BRAF^V600E gene the following formula was used: = [2^(Ct input – Ct isolation)]%