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 may not provide a durable signal. Sequence-specific covalent cross-link formation can anchor probes to target DNA and also may provide an additional layer of target selectivity. Here we develop a new cross-linking reaction for the covalent capture of specific nucleic acid sequences. This process involves reaction of an abasic 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 are easily prepared and display exquisite specificity for the mutant sequence under isothermal assay conditions. DNA duplexes generated in these studies were quantitatively measured using both denaturing gel electrophoresis and a protein nanopore.