Development of An Innovative TEE Technology for Mutation Detection

摘要 该项目的目标是进一步发展一种突破性的立足点-浓缩， 提取（TEE）技术，并验证基于TEE的测试用于癌症检测和护理 管理TEE是一种新的DNA提取方法。与传统的提取方法不同， TEE提取突变的DNA，同时以高回收率富集它。此外，与目前的PCR不同， 基于富集方法，TEE富集突变的DNA而不改变原始DNA序列。 我们已经表明，TEE可以富集突变的DNA多达1000倍。所以当 TEE用于提取DNA进行突变检测试验，它显著增加了分析 测试的灵敏度，导致在低得多的浓度下准确检测突变的DNA 水平，这是如果使用常规方法提取DNA无法实现的。为了举例说明， 当使用常规方法提取DNA时，桑格测序不能检测突变的 DNA，如果其浓度低于20%。相反，当TEE用于提取突变的DNA时， 它使桑格测序能够检测0.1%的突变DNA。换句话说，桑格测序 当我们将TEE结合到桑格测序中时， (we将这种组合称为TEE-Sanger测序）用于突变分析。此外，我们有 证明TEE-Sanger测序可以检测来自临床标本的突变DNA， FFPE肿瘤组织和血液（血清）样本，表明基于TEE的检测兼容 临床应用。 由于TEE是一种DNA提取方法，它可以取代常规方法提取DNA， 用于突变分析的DNA此外，TEE不仅是一项突破性技术， 该平台可以为不同的临床应用开发各种基于TEE的测试。 受我们初步研究的成功和TEE技术的潜力的启发，我们建议 该SBIR第二阶段项目旨在进一步研究TEE技术，为 其商业化。该项目有四个具体目标。

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.