Development of An Innovative TEE Technology for Mutation Detection

摘要 该项目的目标是进一步发展突破性的立足点--浓缩-- 提取(TEE)技术并验证基于TEE的检测在癌症检测和护理中的使用 管理层。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DNA。此外，与目前的聚合酶链式反应不同， 在浓缩方法的基础上，TEE在不改变原始DNA序列的情况下对突变的DNA进行浓缩。 我们已经证明，TEE可以使突变的DNA丰度高达1000倍。因此，当 TEE用于提取DNA以进行突变检测，它显著提高了分析能力 检测的敏感性，导致在低得多的浓度下准确检测突变的DNA 如果使用常规方法提取DNA，这是无法实现的。为了说明， 当使用常规方法提取DNA时，Sanger测序无法检测到突变 如果DNA的浓度低于20%。相反，当TEE被用于提取突变的DNA时， 它使桑格测序能够检测到0.1%的突变DNA。换句话说，桑格排序 当我们将TEE整合到Sanger测序中时，就会变得像数字PCR或NGS一样敏感 (我们称这种组合为TEE-Sanger测序)用于突变分析。此外，我们还拥有 证明了Te-Sanger测序可以从临床标本中检测到突变的DNA FFPE肿瘤组织和血液(血清)样本，表明基于TEE的检测是兼容的 具有临床应用价值。 由于TEE是一种DNA提取方法，可以取代传统的DNA提取方法 用于突变分析的DNA。此外，TEE不仅是一项突破性的技术，而且是一种 可为不同的临床应用开发各种基于TEE的测试的平台。 受我们初步研究的成功和TEE技术潜力的启发，我们建议 本次SBIR二期项目将进一步研究TEE技术，为 它的商业化。这个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.