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The sensitivity of available tests is, however, insufficient for patients at early disease stage and for cancer screening. Detection of alleles that contain critical clinical information is challenged by their very low concentrations and sequence homologies with abundant wildtype nucleic acids. This invention comprises of a high efficiency assay that reduces the concentrations of wildtype nucleic acids, DNA and/or RNA, and increases the fraction of clinically-relevant mutant alleles (DNA and/or RNA) to enable their downstream detection by a variety of methods.
This invention has many advantages over competing technologies such as CRISPR Cas9-based, as Ago does not require targets to contain any specific (PAM-like) motifs; is a multi-turnover enzyme; cleaves ssDNA, dsDNA, and RNA targets in a single assay; is DNA-guided (less expensive and more stable than RNA guides), and operates at elevated temperatures, providing high selectivity and compatibility with polymerases.