Measurements of BPDE-DNA adducts by solid state nonopore and deep sequencing (PQ

通过固态非孔和深度测序 (PQ

• 批准号: 8534070
• 负责人: Rashid Bashir
• 金额: $ 18.8万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-20 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8534070
• 关键词: Air; Area; Benzene; Benzo(a)pyrene; Binding; Bioinformatics; Biological; Biological Markers; Biological Monitoring; Caliber; Cancer Etiology; Carcinogens; Cell Line; Cells; Complex; CpG dinucleotide; DNA; DNA Adducts; DNA Footprint; Detection; Development; Engineering; Epidemiologic Studies; Epigenetic Process; Epoxy Compounds; Equipment; Esophageal; Exposure to; Food; Gel; Genome; Genome Mappings; Genomics; Goals; Guanine Nucleotides; Human; International Agency for Research on Cancer; Investigation; Knowledge; Leukocytes; Link; Lung; Malignant Neoplasms; Maps; Measurement; Measures; Methodology; Methods; Molecular Biology; Mutation; Operative Surgical Procedures; Outcomes Research; Population; Principal Investigator; Procedures; Protein p53; Protocols documentation; Radioactive; Reaction; Reagent; Reporting; Research; Resolution; Risk; Role; Sampling; Scientist; Smoke; Speed; System; Techniques; Testing; Tissues; Tobacco smoke; Toxicology; Toxin; adduct; base; cancer initiation; cancer risk; deep sequencing; design; improved; multidisciplinary; nanopore; nanoscale; novel; programs; ras Oncogene; research study; sensor; single molecule; solid state; tool

Measurements of BPDE-DNA adducts by solid state nanopore and deep sequencing (PQ3) Potent carcinogen Benzo[a]pyrene (BaP) is found in heavily polluted air, smoked food, and tobacco smoke. Once inside cells, BaP can form stable benzo[a]pyrene dihydrodiol epoxide (BPDE) DNA adducts leading to the insertion of incorrect bases during replication. BPDE-DNA adducts are considered biomarkers of exposure to BaP and increased concentrations of BPDE-DNA adducts in white blood cells and in tissues such as lung and esophageal are associated with increased risk of cancer. Current methods for BPDE adduct measurements either involve highly radioactive reagents and are non-specific, or require highly specialized equipment and prohibitively large amounts of DNA for most epidemiological studies. Novel sensors for specific biomonitoring of genomic BPDE adduct which do not require hazardous reagents, or cumbersome procedures will speed the links between BaP exposure and development of various cancers. Furthermore, it is difficult to assess the associations of adduct at specific genomic loci and various cancers. The binding of BPDE on the genomic DNA is not random. Recent reports have demonstrated a preferential distribution of the BPDE-DNA complexes on the methylated CpG dinucleotides and on the mutational hotspots in tumor suppressor p53 and ras oncogene. However, with the existing methodologies BPDE adducts can only be examined at a very limited number of genomic loci. Such investigations would require laborious techniques involving highly radioactive reagents and sequencing gels. Novel non-intensive methodologies for estimating BPDE concentrations on the genome could provide new tools for identifying potential links between BaP exposure and specific mutations and epigenetic alterations that cause cancer. The objective of this proposal is to develop new solid state sensors and novel "omic" -style methodologies for the measurement of DNA adducts. Both approaches will be specifically tuned to BPDE-DNA measurements and will have the sensitivity needed for subsequent studies in human cancer using surgical tissues without requiring prohibitively large amounts of DNA. Aim 1 will develop a solid state nanopore measurement system for single molecule detection of BPDE-DNA adducts. Aim 2 will develop a protocol for genome mapping BPDE adducts in DNA from cell lines exposed to BaP or BPDE. This research will provide the "proof of concept" and scientific evidence upon which subsequent experiments can be designed for assessing BaP exposures in specific populations. Achieving these goals will open new areas of research and provide valuable new tools for fast and detailed measurements of long and short term exposure to the BaP. The approaches introduced here can be extended to most other toxins and carcinogens that form DNA adducts. These findings will benefit diverse areas of cancer and scientific research, such as toxicology, where adducts are studied.

通过固态纳米孔和深度测序对BPDE-DNA加合物的测量（PQ3） 在严重污染的空气，烟熏食品和烟草烟雾中发现了有效的致癌苯并[A] pyrene（BAP）。一旦在细胞内部，BAP会形成稳定的苯并[A] pyrene二氢二醇环氧（BPDE）DNA加合物，导致复制过程中插入不正确的碱基。 BPDE-DNA加合物被认为是暴露于BAP的生物标志物和白细胞中BPDE-DNA加合物的浓度增加，以及肺和食管等组织中的BPDE-DNA加合物与癌症的风险增加有关。当前的BPDE加合物测量方法涉及高放射性试剂，并且是非特异性的，或者需要高度专业化的设备，并且对于大多数流行病学研究而言，大量的DNA大量DNA。不需要危险试剂的基因组BPDE加合物特异性生物监测的新型传感器，或者繁琐的程序将加快BAP暴露与各种癌症的发展之间的联系。此外，很难评估特定基因组基因座和各种癌症的加合物的关联。 BPDE对基因组DNA的结合不是随机的。最近的报道表明，BPDE-DNA复合物在甲基化的CpG二核苷酸以及肿瘤抑制p53和Ras癌基因中的突变热点上的优先分布。但是，使用现有的方法论，BPDE加合物只能以非常有限的基因组基因座进行检查。这样的研究将需要涉及高放射性试剂和测序凝胶的费力技术。用于估计基因组BPDE浓度的新型非密集型方法可以提供新的工具，以识别BAP暴露与特定突变与引起癌症的表观遗传改变之间的潜在联系。该建议的目的是开发新的固态传感器和新型的“ OMIC”式方法，用于测量DNA加合物。两种方法都将专门调整为BPDE-DNA测量结果，并将使用手术组织对人类癌症进行随后研究所需的敏感性，而无需大量的大量DNA。 AIM 1将开发一个固态纳米孔测量系统，用于单分子检测BPDE-DNA加合物。 AIM 2将从暴露于BAP或BPDE的细胞系中开发DNA中基因组映射BPDE加合物的方案。这项研究将提供“概念证明”和科学证据，以此为其设计用于评估特定人群中BAP暴露的后续实验。实现这些目标将开放新的研究领域，并为长期和短期暴露于BAP的快速和详细测量提供宝贵的新工具。此处引入的方法可以扩展到形成DNA加合物的大多数其他毒素和致癌物。这些发现将使研究合并的癌症和科学研究的各个领域，例如毒理学。