High Quality, High Integrity Nucleic Acid Extraction from FFPE Tissues

点击翻译按钮获取中文摘要

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.