Nanopore Detection of DNA Damage

描述(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碱基经历了持续的修改。DNA测序技术不能直接检测DNA损伤，因为测序是在PCR扩增的链上进行的，这些链必须包含4个典型的碱基A、C、T和G。通过测序可以检测到突变，其中许多突变是DNA损伤的最终结果。然而，突变本身并不能提供有关原始损伤的化学特性的太多信息。该项目将研究一种检测DNA碱基修饰(如氧化、烷基化或切除)的方法，方法是应用化学和酶方法将修饰的碱基转化为加合物，当单个DNA链通过膜嵌入的离子通道时，加合物会产生可检测的信号。这种方法将提供对单分子DNA损伤的直接读出，长期目标是开发与微流控相兼容的方法来分析来自细胞来源的非常少量的DNA样本。该项目的具体目标是：(1)通过有机和酶化学的结合，优化特定DNA损伤到纳米孔离子通道方法可检测到的加合物的转化；(2)优化离子通道测量，以检测和定量单部位DNA损伤，并证明携带加合物的DNA链通过孔转运；(3)验证化学损伤后使用大DNA靶点(例如，质粒M13mp18)的方法；以及(4)开发一种通过产生特定的5dNTP来扩大DNA损伤的方法，用于酶标记损伤部位。该项目长期目标的实现将影响人类健康研究的三个领域：(1)个性化药物治疗，(2)疾病的早期发现，(3)表观遗传学。

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.