Nanopore Detection of DNA Damage

DNA损伤的纳米孔检测

• 批准号: 8323314
• 负责人: Cynthia J Burrows
• 金额: $ 28.6万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 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/8323314
• 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

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.

描述（由申请人提供）：由于引入表观遗传标记物以及通过内源性源或毒性的氧化和烷基化反应引入表观遗传标志物以及暴露于DNA损害剂的自然过程，基因组和线粒体DNA碱基都经历了连续的修饰。 DNA测序技术不能直接检测到DNA损伤，因为测序发生在PCR放大的链接上仅包含4个规范碱基A，C，T和G。可以通过测序检测突变，其中许多是DNA损伤的最终结果。但是，突变本身并没有提供有关原始损害的化学身份的太多信息。该项目将通过应用化学和酶法方法检测DNA碱基修饰（例如氧化，烷基化或切除）的方法，将修饰的碱转换为加合物，当通过膜上包含的离子通道转移单个DNA链时，该加合物将产生可检测的信号。该方法将直接读取单分子对DNA损伤的长期目标是开发与微流体兼容的方法，以分析来自细胞来源的非常小的DNA样品。该项目的具体目的是（1）优化特定的DNA病变通过有机和酶促化学的组合通过纳米孔离子通道方法检测到的加合物的转化，（2）优化离子通道测量值，以检测和量化单点DNA损伤，并证明使用DNA链质量的范围（3）通过了dna cortrants tallains tallacte（3）。由于化学损伤后质粒M13MP18，并且（4）通过生成特定的第5个DNTP来扩大DNA损伤的方法，以促进损伤位点的酶促划分。实现该项目的长期目标将影响3个领域的人类健康研究：（1）个性化药物治疗，（2）早期疾病的早期发现和（3）表观遗传学。