Nanopore Detection of DNA Damage

DNA损伤的纳米孔检测

基本信息

批准号：
8040859
负责人：
Cynthia J Burrows
金额：
$ 27.06万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8040859
关键词：
Alkylation Area Binding Caliber Caring Cells Chemicals Chemistry DNA DNA Adducts DNA Damage DNA Modification Process DNA Repair Enzymes DNA Sequence DNA lesion Deamination Dependence Detection Disease Early Diagnosis Enzymatic Biochemistry Epigenetic Process Evaluation Excision Exposure to Generations Genes Genetic Genome Genomics Glass Goals Health Hemolysin Hot Spot Human Human Genome Individual Ion Channel Lesion Link Lipid Bilayers Measurement Membrane Methodology Methods Microfluidics Mitochondrial DNA Modification Monitor Mutation Outcome Pharmacotherapy Physicians Plasmids Process Reaction Reading Research Sampling Shapes Signal Transduction Single-Stranded DNA Site Source Streptavidin Structure Surface Techniques Tissue Sample Toxic Environmental Substances Uracil adduct analytical method base cancer therapy fluorescence imaging nanopore oxidation plasmid DNA porin single molecule tool toxicant

项目摘要

DESCRIPTION (provided by applicant): Genomic and mitochondrial DNA bases undergo continuous modifications as a result of both natural processes that introduce epigenetic markers as well as exposure to DNA damaging agents through oxidation and alkylation reactions from endogenous sources or toxicants. DNA sequencing techniques do not directly detect DNA damage because the sequencing takes place on PCR-amplified strands that perforce contain only the 4 canonical bases A, C, T, and G. Mutations can be detected by sequencing, and many of these are the ultimate outcome of DNA damage. However, mutations themselves do not provide much information about the chemical identity of the original damage. This project will examine an approach to detection of DNA base modification (e.g. oxidation, alkylation, or excision) by application of chemical and enzymatic methods to convert the modified base to an adduct that yields a detectable signal when individual DNA strands translocate through a membrane-embedded ion channel. This method will provide a direct read-out of DNA damage on single molecules The long-term goal is to develop methodology compatible with microfluidics to analyze very small samples of DNA from cellular sources. The specific aims of this project are to (1) optimize the conversion of specific DNA lesions to adducts detectable by the nanopore ion channel method by a combination of organic and enzymatic chemistries, (2) optimize the ion channel measurements to detect and quantify single-site DNA damage and demonstrate that DNA strand carrying adducts are translocated through the pore, (3) validate the methods using large DNA targets such as the plasmid M13mp18 after chemical damage, and (4) develop a method to PCR amplify DNA damage by generation of a specific 5th dNTP for enzymatic demarcation of damage sites. Realization of the long-term goals of this project will impact research in human health in 3 areas: (1) personalized drug therapy, (2) early detection of disease, and (3) epigenetics. PUBLIC HEALTH RELEVANCE: The ability to monitor specific DNA base damage sites in tissue samples would have a significant impact on human health by rapid evaluation of genetic damage caused by cancer treatments or environmental toxins. In addition, the ability to detect DNA damage occurring in specific genes may guide physicians to preventative care. The methods developed in this research may also provide epigenetic tools for linking DNA modification to disease states.

描述（由申请人提供）：由于引入表观遗传标记的自然过程以及通过内源性来源或有毒物质的氧化和烷基化反应暴露于 DNA 损伤剂，基因组和线粒体 DNA 碱基经历持续修饰。 DNA 测序技术不能直接检测 DNA 损伤，因为测序是在 PCR 扩增的链上进行，而这些链必然只包含 4 个典型碱基 A、C、T 和 G。突变可以通过测序来检测，其中许多突变是最终的结果。 DNA损伤的结果。然而，突变本身并不能提供有关原始损伤化学特性的太多信息。该项目将研究一种检测 DNA 碱基修饰（例如氧化、烷基化或切除）的方法，通过应用化学和酶方法将修饰的碱基转化为加合物，当单个 DNA 链通过膜易位时，加合物会产生可检测的信号。嵌入离子通道。该方法将直接读出单分子上的 DNA 损伤。长期目标是开发与微流体兼容的方法来分析来自细胞来源的非常小的 DNA 样本。该项目的具体目标是（1）通过有机和酶化学的组合，优化特定 DNA 损伤到可通过纳米孔离子通道方法检测的加合物的转化，（2）优化离子通道测量以检测和量化单-位点 DNA 损伤并证明携带加合物的 DNA 链通过孔易位，(3) 验证化学损伤后使用大 DNA 靶标（例如质粒 M13mp18）的方法，以及 (4) 开发 PCR 方法通过生成特定的第 5 个 dNTP 来放大 DNA 损伤，以酶法划分损伤位点。该项目长期目标的实现将影响三个领域的人类健康研究：(1)个性化药物治疗，(2)疾病早期检测，(3)表观遗传学。公共健康相关性：通过快速评估癌症治疗或环境毒素引起的遗传损伤，监测组织样本中特定 DNA 碱基损伤位点的能力将对人类健康产生重大影响。此外，检测特定基因中发生的 DNA 损伤的能力可以指导医生进行预防性护理。本研究开发的方法还可能提供将 DNA 修饰与疾病状态联系起来的表观遗传学工具。