Info

Attempted single-molecule classifications

FIGURE 11.11 Classification of a 3:1 mixture of 9 bp TA and 9 bp GC hairpin molecules as a function of single-molecule acquisitions. The proportion of 9 bp TA was identified with 99.9% accuracy within 100 events. (From Winters-Hilt, S., Vercoutere, W., DeGuzman, V., Deamer, D., Akeson, M., and Haussler, D., Biophys. J., 87, 967, 2003. With permission.)

Attempted single-molecule classifications

FIGURE 11.11 Classification of a 3:1 mixture of 9 bp TA and 9 bp GC hairpin molecules as a function of single-molecule acquisitions. The proportion of 9 bp TA was identified with 99.9% accuracy within 100 events. (From Winters-Hilt, S., Vercoutere, W., DeGuzman, V., Deamer, D., Akeson, M., and Haussler, D., Biophys. J., 87, 967, 2003. With permission.)

proposed to be a reversible unzipping step of the DNA up to the 4 bp mismatch, followed by the second rate constant representing a much slower unzipping of the remaining base pairs.

Mathe et al.22 applied a controlled voltage ramp to draw DNA through the pore, and measured the duration that the molecule remained double-stranded, in order to calculate the force required to initiate unzipping. In these experiments, DNA capture is rapidly followed by a drop in the potential to a holding force; then ramping from 0.5 to 100 V/s is performed. The results from this method showed agreement to other single-molecule measurements of DNA unzipping rates at high loading rates, but a weaker dependence at lower rates (<5 V/s). This new approach provides a means to test DNA unzipping at forces lower than accessible by, for example, optical tweezers or atomic force microscopy.

Both Sauer and Mathe used voltage-dependent rate constants for DNA unzipping to estimate the effective charge of a nucleotide in the pore at ~0.1e. This low value maybe explained by charge shielding from the counterion distribution along the negatively charged DNA backbone.

0 0

Post a comment