Nanopore Detection of DNA Damage

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毒物的氧化和烷基化反应暴露于DNA损伤剂，基因组和线粒体DNA碱基经历连续修饰。DNA测序技术不能直接检测DNA损伤，因为测序发生在PCR扩增的链上，这些链必然只含有4种典型碱基A、C、T和G。突变可以通过测序检测到，其中许多是DNA损伤的最终结果。然而，突变本身并不能提供关于原始损伤的化学身份的太多信息。本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，通过应用化学和酶促方法将修饰的碱基转化为加合物，当单个DNA链通过膜嵌入的离子通道移位时，该加合物产生可检测的信号。这种方法将提供单分子DNA损伤的直接读出。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。该项目的具体目标是（1）通过有机和酶化学的组合，优化特定DNA损伤向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链通过孔移位，（3）在化学损伤后使用大的DNA靶标如质粒M13mp18验证方法，和（4）开发通过产生特异性第5 dNTP用于损伤位点的酶划界来PCR扩增DNA损伤的方法。该项目长期目标的实现将影响3个领域的人类健康研究：（1）个性化药物治疗，（2）疾病的早期检测，和（3）表观遗传学。 公共卫生相关性：监测组织样本中特定DNA碱基损伤位点的能力将通过快速评估癌症治疗或环境毒素造成的遗传损伤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。这项研究中开发的方法也可能提供将DNA修饰与疾病状态联系起来的表观遗传工具。

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.