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- W3132833648 abstract "Genomic single-nucleotide variants (SNVs) can result in significant functional consequences to an organism and give rise to diseases such as cancer. As a result, the ability to detect rare DNA mutations in biofluids is a crucial diagnostic tool for the early detection of disease. We outline a strategy for detecting SNVs in DNA sequences against a large background of similar sequences using the principles of kinetic proofreading. Kinetic proofreading is a natural mechanism by which an incorrect substrate is selectively removed from a chemical reaction pathway through one or more irreversible “exit” reactions, thereby achieving a multiplicative increase in molecular specificity with each additional exit step. We propose a kinetic proofreading system that uses repeated toehold-mediated DNA strand displacement (DSD) to detect SNVs in DNA sequence with extremely high specificity. Kinetic proofreading with DSD works via competitive hybridization of DNA probes to SNV-containing target sequences followed by selective liberation of the target toehold. This process can be performed iteratively, where the target is captured by mutant-selective probes and wildtype sequences are ejected from the reaction pathway via irreversible hybridization with wildtype-selective probes. The target is freed after each round through re-exposure of the toehold. Early investigations suggest that using DNA:RNA chimeric duplex probes and RNase H is a reliable method for doing this. While the discrimination of each round of proofreading is finite, it can be repeated on the same pool of molecules, permitting theoretically unlimited selectivity as the number of iterations increases. This contrasts to the strictly finite selectivity of conventional hybridization assays, such as quantitative polymerase chain reaction (qPCR). This work will represent a new capability for molecular recognition, which we predict will bypass the thermodynamic limits of discrimination of DNA hybridization at equilibrium and may achieve virtually unlimited selectivity." @default.
- W3132833648 created "2021-03-01" @default.
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- W3132833648 date "2021-02-01" @default.
- W3132833648 modified "2023-10-18" @default.
- W3132833648 title "Iterative Kinetic Proofreading for High-Specificity DNA Sequence Discrimination" @default.
- W3132833648 doi "https://doi.org/10.1016/j.bpj.2020.11.1724" @default.
- W3132833648 hasPublicationYear "2021" @default.
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