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Microfluidics-assisted display of stretched DNA in the study of DNA repair in viv

体内 DNA 修复研究中微流控辅助显示拉伸 DNA

基本信息

批准号：
8265953
负责人：
JULIA SIDOROVA
金额：
$ 15.6万
依托单位：
UNIVERSITY OF WASHINGTON
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：
2011-03-01 至 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8265953
关键词：
Adopted Base Excision Repairs Biological Assay Biological Models Cells Chromosomal Rearrangement Clinical DNA DNA Damage DNA Repair DNA Repair Gene DNA analysis DNA biosynthesis Data Defect Diagnostic Disease Ear Epidemiologic Studies Epigenetic Process Fibroblasts Future Gene Expression Profile Genetic Polymorphism Genetic Variation Genome Genome Stability Genotype Goals Heterozygote Human Inherited Knowledge Lead Life Lymphocyte Malignant Neoplasms Measures Methods Methyl Methanesulfonate Methylation Microfluidics Modeling Mus Mutagens Mutation Organism Outcome Patients Performance Peripheral Blood Mononuclear Cell Physiological Process Proteins Research Research Design Resolution Risk Sampling Single Nucleotide Polymorphism Source Specimen Staging Stretching System Technology Testing Therapeutic UV induced DNA damage Variant Work XRCC1 gene adduct base cancer risk carcinogenesis clinically relevant disorder risk dosage epigenetic variation human XRCC1 protein in vivo lymphoblastoid cell line repaired response tool tumor

项目摘要

DESCRIPTION (provided by applicant): The goal of the proposed research is to develop and apply a new quantitative method of assaying DNA repair efficiency in vivo, suitable for diagnostic use in readily available patient material. Genomic stability of living cells is continuously threatened from without, by environmental genotoxins as well as from within, by reactive metabolites and enzymatic malfunction. Cells have evolved multiple mechanisms to repair a broad spectrum of damages to DNA, and it is generally thought that small genetic or epigenetic variations in the proficiency of DNA repair may have a profound lifetime impact on genomic stability of an organism. Recent years have seen an exponential increase in studies that connect DNA repair gene single nucleotide polymorphisms (SNPs), expression levels, or methylation status to cancer risk or therapeutic outcome. Understanding the functional, mechanistic significance of the genotypic, epigenetic, or transcriptome signatures found associated with disease risk should deepen our knowledge of the disease process, validate association studies and, ultimately, inform future clinical decisions. In the past several years I have adopted a high resolution, global DNA analysis tool - microfluidics-assisted display of stretched DNA molecules - and used it for a quantitative analysis of DNA replication in vivo and its response to DNA damage in human cells. In this proposal I aim to adapt this technology to a new, thus far unrealized application -- to measure DNA repair in vivo. I will develop, validate, and apply an adaptation of our DNA-stretching technology to study DNA repair using base excision repair (BER) of methyl adducts in mouse primary fibroblasts as a model system. Using cells with dosage or mutation defects in XRCC1, a protein critical for BER, I will determine whether my approach allows measuring relatively small variations in BER efficiency. At the next stage, I will test whether I can apply my technology to measure DNA repair in clinically relevant samples such as human peripheral blood mononuclear cells and human lymphoblastoid cell lines homozygous for the SNPs in XRCC1 that are associated with increased risk of cancer. Finally, I will take steps to determine applicability of my technology to the study of DNA repair systems other than BER.

描述(由申请人提供)：拟议研究的目标是开发和应用一种新的定量方法来检测体内DNA修复效率，适用于在容易获得的患者材料中的诊断使用。活细胞的基因组稳定性不断受到来自外部、环境遗传毒素以及内部、反应性代谢物和酶故障的威胁。细胞已经进化出多种机制来修复广泛的DNA损伤，通常认为DNA修复熟练程度上的微小遗传或表观遗传差异可能会对生物体的基因组稳定性产生深远的影响。近年来，将DNA修复基因单核苷酸多态性(SNPs)、表达水平或甲基化状态与癌症风险或治疗结果联系起来的研究呈指数级增长。了解与疾病风险相关的遗传型、表观遗传学或转录组特征的功能和机制意义，应该加深我们对疾病过程的知识，验证关联研究，并最终为未来的临床决策提供信息。在过去的几年里，我采用了一种高分辨率的全球DNA分析工具-微流体辅助显示拉伸的DNA分子-并使用它对体内DNA复制及其对人类细胞DNA损伤的反应进行了定量分析。在这项提议中，我的目标是将这项技术应用于一种新的、迄今尚未实现的应用--测量体内DNA修复。我将开发、验证并应用我们的DNA拉伸技术来研究DNA修复，以小鼠原代成纤维细胞中甲基加合物的碱基切除修复(BER)为模型系统。利用XRCC1(一种对误码率至关重要的蛋白质)中存在剂量或突变缺陷的细胞，我将确定我的方法是否允许测量误码率效率的相对较小的变化。在下一阶段，我将测试是否可以应用我的技术来测量临床相关样本的DNA修复，例如人类外周血单核细胞和人类淋巴母细胞系，XRCC1中的SNP纯合与癌症风险增加相关。最后，我将采取措施确定我的技术是否适用于BER以外的DNA修复系统的研究。