Figure 4.

Cross-linked DNA generated from the mutant BRAF-probe duplex E can be readily detected by its unique current signature in the α-HL nanopore. The mixtures of species generated by incubation of the third-generation probe with either mutant or wild-type BRAF gene sequence were analyzed using a single α-HL ion channel embedded in a lipid bilayer. Current traces were recorded at +120 mV in Tris (10 mM, pH 7.4) containing KCl (1 M) at 22 ℃. Panel A: Analysis of the mixture generated by hybridization of the mutant BRAF sequence with the Ap-containing probe strand. The current block was recorded for 1 min, then voltage polarity was reversed to translocate the cross-linked duplex back to the cis solution. Current trace showing persistence of the current block by cross-linked duplex E for 30 min is provided in Figure S6. Panel B: The wild-type BRAF sequence does not generate cross-linked DNA when hybridized with the Ap-containing probe strand. Short current blocks are consistent with translocation of single-stranded DNA and uncross-linked duplex F. The illustration depicts the three-step unzipping/translocation process for duplex DNA.