BEAMing

Purified DNA of PDX mouse model tumors was sent to Sysmex Inostics, Hamburg, Germany. DNA content was quantified using a modified version of LINE-1 Real-time PCR as previously described [48, 49]. An amount of 330 ng DNA per sample was inputted for each tissue BEAMing analysis. BEAMing digital PCR is a technique in which individual DNA molecules are attached to magnetic beads in water-in-oil emulsions and then subjected to compartmentalized (digital) PCR amplification [50]. The mutational status of DNA bound to beads was then determined by hybridization of fluorescently labeled allele-specific probes for mutant or wildtype KRAS G12V (g35t) sequences. Finally, the bead population was analyzed by flow cytometry to count and sort wildtype and mutant beads. The result is reported as the fractional abundance of mutant DNA alleles relative to wildtype DNA alleles per sample. To generate the ratio of mutant to wildtype DNA alleles (mutant allelic fraction, MAF), an average of 3 × 10⁶ beads were interrogated in each BEAMing analysis (approximately 90,000 beads per mutation). The cut-off for calling a mutation in xenograft derived DNA with BEAMing was set to 0.02% MAF.