Optimized digital polymerase chain reaction enables detection of telomerase reverse transcriptase C228T mutation for prognostic assessment in hepatocellular carcinoma

Figure 2 Two-dimensional digital polymerase chain reaction plots showing the telomerase reverse transcriptase C228T promoter assay with and without 7-deaza-dGTP, CviQI and EDTA reagents in patient HC2873. A: Preliminary results of amplification and detection of the C228T fragment using standard supermixtures and assays (left); the detailed amount of each reagent (right); B: The optimized detection conditions, including 7-deaza-DGTP, CviQI and EDTA reagents, and the experimental results of generating distinct droplet clusters for a C228T fragment (left). The optimized detailed dosage of each reagent (right). Color of line: Green, VIC probe telomerase reverse transcriptase (TERT) C228T wild-type; dark blue, 6-carboxyfluorescein probe for TERT C228T mutant type; light blue, droplets with TERT C228T mutant and wild-type allele; dark grey, droplets negative for template DNA. The green and blue lines are manually set thresholds for a 6-carboxyfluorescein mutant probe and VIC wild-type probe for digital polymerase chain reaction mutation detection, respectively.