Sequence-Specific Covalent Capture Coupled with High-Contrast Nanopore Detection of a Disease-Derived Nucleic Acid Sequence.

Hybridization-based methods for the detection of nucleic acid sequences are important in research and medicine. Short probes provide sequence specificity, but do not always provide a durable signal. Sequence-specific covalent crosslink formation can anchor probes to target DNA and might also provide an additional layer of target selectivity. Here, we developed a new crosslinking reaction for the covalent capture of specific nucleic acid sequences. This process involved reaction of an abasic (Ap) site in a probe strand with an adenine residue in the target strand and was used for the detection of a disease-relevant T→A mutation at position 1799 of the human BRAF kinase gene sequence. Ap-containing probes were easily prepared and displayed excellent specificity for the mutant sequence under isothermal assay conditions. It was further shown that nanopore technology provides a high contrast-in essence, digital-signal that enables sensitive, single-molecule sensing of the cross-linked duplexes.