3 years ago

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

Sequence-Specific Covalent Capture Coupled with High-Contrast Nanopore Detection of a Disease-Derived Nucleic Acid Sequence
Ruicheng Shi, Maryam Imani Nejad, Xinyue Zhang, Kent S. Gates, Li-Qun Gu
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 TA 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. Reaching out and grabbing hold of specificity: Interstrand crosslinking chemistry can be used for highly selective covalent anchoring of a probe to a specific nucleic acid target sequence. We developed a new crosslinking reaction that uses probes prepared in a one-step procedure from inexpensive commercial reagents to achieve excellent sequence specificity under isothermal assay conditions.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/cbic.201700204

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