Development of An Innovative TEE Technology for Mutation Detection

Abstract The objective of this project is to further develop a breakthrough Toehold-Enrichment- Extraction (TEE) technology and validate TEE-based tests for use in cancer detection and care management. TEE is a novel DNA extraction method. Unlike conventional extraction methods, TEE extracts mutated DNA while enriching it with high recovery. Moreover, unlike current PCR-

based enrichment methods, TEE enriches mutated DNA without altering original DNA sequences. We have shown that TEE could enrich mutated DNA by as much as 1000 fold. Therefore, when TEE is used to extract DNA for a mutation detection test, it significantly increases analytical sensitivity of the test, leading to accurate detection of mutated DNA at a much lower concentration

level, which could not be achieved if a conventional method is used to extract DNA. To illustrate, when a conventional method is used to extract DNA, Sanger sequencing cannot detect mutated DNA if its concentration is less than 20%. In contrast, when TEE is used to extract mutated DNA, it enables Sanger sequencing to detect 0.1% mutated DNA. In other words, Sanger-sequencing

becomes as sensitive as digital PCR or NGS when we incorporate TEE into Sanger sequencing (we term this combination TEE-Sanger sequencing) for mutation analysis. Furthermore, we have demonstrated that TEE-Sanger sequencing could detect mutated DNA from clinical specimen like FFPE tumor tissue and blood (serum) samples, indicating that TEE-based testing is compatible

with clinical applications. Since TEE is a DNA extraction method, it can replace conventional methods to extract DNA for mutation analysis. Moreover, TEE is not only a breakthrough technology, but also a platform on which various TEE-based tests can be developed for different clinical applications. Inspired by the success of our preliminary study and potential of the TEE technology, we propose

this SBIR Phase II project to further study the TEE technology, providing a strong foundation for its commercialization. This project has four specific aims.