High Quality, High Integrity Nucleic Acid Extraction from FFPE Tissues

项目摘要 福尔马林固定石蜡包埋（FFPE）组织样本已发展成为基因组学的宝贵资源 研究并成为许多分子癌症测试的实际样本类型。虽然FFPE处理可以 为了充分稳定核酸以用于运输和储存，现有的提取方法难以获得高的提取率。 由于交联、断裂和有机污染，DNA/RNA质量下降。在第一阶段，我们 使用Nanobind从各种新鲜和固定的DNA中提取高质量DNA的可行性证明研究 组织样本Nanobind是一种新型热塑性纳米材料，可以廉价制造， 能够提取比任何竞争方法更高质量的DNA。目前的提取方法 经过努力，我们已经证明，使用Nanobind可以获得极高的质量， 来自新鲜FFPE样品的分子量DNA（100 kb+）。另外，我们发现紫外光谱法， Qubit/PicoGreen分析和电泳提供了一个不完整的DNA质量的图片，往往是穷人 测序和基因组作图中的性能预测因子。损伤病变，如刻痕，脱碱基位点， FFPE样品中普遍存在的蛋白质-DNA交联和DNA-DNA交联不能通过 这些方法。这种损伤首先在固定期间产生，然后在随后的固定期间复合。 存储和苛刻的提取过程。由于缺乏合适的测定来量化各种损伤病变， 关于预分析和样品制备因素如何影响DNA质量，除了它们的 对产量、总杂质和完整性的影响。在第二阶段，我们会在第一阶段研究的基础上， 新的检测方法来量化特定的DNA损伤病变，然后使用这些检测方法来完善我们对 FFPE样品处理。首先，我们将开发简单的荧光分析来量化3种常见形式的DNA 损伤：刻痕、脱碱基位点和脱氨基。第二，我们将利用这些检测方法进一步改善 从FFPE组织样本中提取的DNA的质量，并研究FFPE的上游效应 处理损伤病灶。最后，我们将通过以下方式验证Nanobind FFPE DNA提取性能： 从新鲜、固定和加速老化样品中分离DNA，使用标准品 方法和新开发的DNA损伤检测方法，并与NGS和长读 测序数据。

Project Summary Formalin-fixed paraffin embedded (FFPE) tissue samples have evolved into a valuable resource for genomics research and become a de facto sample type for many molecular cancer tests. While FFPE processing can adequately stabilize nucleic acids for transport and storage, existing extraction methods struggle to obtain high quality DNA/RNA due to cross-linking, fragmentation, and organic contamination. In Phase I, we performed proof-of-feasibility studies using Nanobind to extract high quality DNA from various types of fresh and fixed tissue samples. Nanobind is a novel thermoplastic nanomaterial that can be inexpensively manufactured and is capable of extracting higher quality DNA than any competing method. Where current extraction methods have struggled, we have demonstrated that with Nanobind it is possible to obtain extremely high quality, high molecular weight DNA (100 kb+) from fresh FFPE samples. Separately, we discovered that UV spectrometry, Qubit/PicoGreen assays, and electrophoresis provide an incomplete picture of DNA quality and are often poor predictors of performance in sequencing and genome mapping. Damage lesions, such as nicks, abasic sites, protein-DNA crosslinks, and DNA-DNA crosslinks, that are pervasive in FFPE samples cannot be detected by these methods. Such damage is first generated during fixation and then compounded during subsequent storage and harsh extraction processes. Due to the lack of suitable assays to quantify various damage lesions, little is known about how preanalytical and sample preparation factors impact DNA quality other than their effects on yield, gross impurities, and integrity. In Phase II, we will build upon our Phase I studies to develop new assays to quantify specific DNA damage lesions and then use these assays to refine our understanding of FFPE sample processing. First, we will develop simple fluorescent assays to quantify 3 common forms of DNA damage: nicks, abasic sites, and deamination. Second, we will utilize these assays to further improve the quality of Nanobind extracted DNA from FFPE tissue samples and to study the upstream effects of FFPE processing on damage lesions. Finally, we will validate Nanobind FFPE DNA extraction performance by isolating DNA from fresh, fixed, and accelerated aging samples, characterizing the DNA using standard methods and the newly developed DNA damage assays, and comparing against NGS and long-read sequencing data.