Figure 2.

Different CSF3R mutations and their initial responses to treatment. CSF3R exons 14 and 17 were amplified from total peripheral blood leukocyte DNA and Sanger sequenced.⁵ To quantify the level of the T618I mutation, we designed a droplet digital PCR (ddPCR) assay. The ddPCR reaction mixture consisted of 10μl ddPCR supermix (Bio-Rad, Hemel Hempstead, UK), 250nm wild type probe ([HEX]ACCC[T]GA[T]GA[C]CT[T]GACC[BHQ1]), and mutation specific probe ([6FAM]ACCC[T]GA[T]GA[T]CT[T]GACC[BHQ1]), 900nm forward (CCACCAACAGTACAGTCC) and reverse (ACCAGGGGATTCAAAGTC) primers, and 7.2μl of fragmented DNA at 16.25ng/μl (bases in square brackets are locked nucleic acids). The entire reaction was loaded into a cartridge (Bio-Rad) together with 70μl droplet generation oil (Bio-Rad) and placed into the droplet generator (Bio-Rad). After processing, the droplets were transferred to a 96 well plate which was sealed with a heat sealer. PCR amplification was performed in a Thermal Cycler (Applied Biosystems 2720) using the following cycling conditions: 95°C for 10 mins, 40 cycles of 94°C for 30s and 60.8C for 1 min, 1 cycle of 98°C for 10 mins and ending at 4°C. After amplification, the plate was loaded onto the droplet reader (Bio-Rad) and analysed immediately with Quantasoft analysis software (Bio-Rad).