Figure 4: SPDBK base discrimination in DNA clusters. | Nature Communications

Figure 4: SPDBK base discrimination in DNA clusters.

From: DNA sequencing using polymerase substrate-binding kinetics

Figure 4

The SS AMP is defined as the point at which the fluorescence response returns to a baseline. For each [NaCl] and [dNTP] data point, the averages of the maximum amplitude (MAX AMP) and steady-state amplitude, correct (SS AMP (C)) are calculated from the three independent correct and mismatch measurements following the synchronization step. The error bars represent the s.d. for each of the conditions calculated from three independent measurements that are recorded on the same clusters at different positions on the calibration template. Since each condition is run on the same flow cell in the same lane, the error bars on the measurement are mostly reflective of differences in fluidic delivery, and potential signal loss due to incomplete extension, misincorporation, or read forward. (a) The peak amplitude correct (red bars) and mismatch nucleotide responses (blue bars) were determined from the raw traces (Supplementary Fig. 3a,b) over a range of NaCl concentrations, 62–375 mM. The maximum fluorescence response from the correct and mismatch traces are ascribed to the extent of synchronized enzyme–DNA binding. The net result was a NaCl-dependent fivefold increase () in correct signal versus mismatch from 62.5 to 375 mM NaCl (b) For higher NaCl concentrations, the MAX AMP (blue) increased, while SS AMP amplitudes (red) decreased. The ratio of the MAX AMP/SS AMP increased approximately fivefold at the highest NaCl concentrations. (c) To find the ideal dNTP concentrations for maximum discrimination power, a range of dNTP concentrations of 5–500 μM were introduced into flow cells with 150 nM labelled BSU polymerase at a fixed NaCl concentration (300 mM). The peak amplitudes of correct (red bars) and mismatch nucleotide responses (blue bars) were determined from the raw traces (Supplementary Fig. 3c,d) over a range of dNTP concentrations from 1 to 500 μM. The net result was a dNTP dependent 5-fold increase () in correct signal versus mismatch at 100 μM. (d) The MAX AMP (blue bars) response peaked around 100 μM while the SS AMP (red bars) increase slightly to yield a fourfold improvement in discrimination at a nucleotide concentration around 50 μM.

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