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High Quality, High Integrity Nucleic Acid Extraction from FFPE Tissues

从 FFPE 组织中提取高质量、高完整性的核酸

基本信息

批准号：
9393293
负责人：
Kelvin Liu
金额：
$ 75.91万
依托单位：
CIRCULOMICS, INC.
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9393293
关键词：
Affect Aging Biological Assay Biological Sciences Biotechnology Capillary Electrophoresis Clinical Complement Cytolysis DNA DNA Damage DNA analysis DNA-protein crosslink Data Deamination Electrophoresis Ensure Feasibility Studies Fluorescence Formalin Freezing Gel Generations Genome Mappings Genomics Heating High temperature of physical object Industrialization Label Lesion Malignant Neoplasms Measurement Methods Molecular Molecular Analysis Molecular Weight Nucleic Acids Nucleotides Output Paraffin Embedding Pathology Performance Phase PicoGreen Preparation Process Protocols documentation RNA Reproducibility Research Resources Sampling Scientist Sepharose Site Sonication Source Spectrometry Systems Analysis Testing Time Tissue Embedding Tissue Sample Tissues UV Radiation Exposure Veterans Xylene base clinical diagnostics crosslink design experience improved nanomaterials next generation sequencing novel organic contaminant phase 1 study prevent qubit sample fixation single molecule tool

项目摘要

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.

项目总结