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（PQ 3）测量BPDE-DNA加合物 苯并[a]芘（BaP）是一种致癌物质，存在于严重污染的空气、烟熏食品和烟草烟雾中。一旦进入细胞，BaP可以形成稳定的苯并[a]芘二氢二醇环氧化物（BPDE）DNA加合物，导致复制过程中插入错误的碱基。BPDE-DNA加合物被认为是苯并（a）芘暴露的生物标志物，白色血细胞以及肺和食管等组织中BPDE-DNA加合物浓度的增加与癌症风险的增加有关。目前用于BPDE加合物测量的方法要么涉及高放射性试剂并且是非特异性的，要么对于大多数流行病学研究需要高度专业化的设备和禁止大量的DNA。用于基因组BPDE加合物的特异性生物监测的新型传感器不需要危险试剂或繁琐的程序，将加速BaP暴露与各种癌症发展之间的联系。此外，很难评估加合物在特定基因组位点和各种癌症的关联。BPDE与基因组DNA的结合不是随机的。最近的研究表明，BPDE-DNA复合物优先分布在甲基化的CpG二核苷酸和肿瘤抑制基因p53和ras的突变热点上。然而，使用现有的方法，BPDE加合物只能在非常有限数量的基因组位点上进行检查。这样的研究需要费力的技术，包括高放射性试剂和测序凝胶。用于估计基因组上BPDE浓度的新的非密集方法可以提供新的工具，用于确定BaP暴露与导致癌症的特定突变和表观遗传改变之间的潜在联系。该提案的目标是开发新的固态传感器和新型“组学”式方法来测量DNA加合物。这两种方法都将专门针对BPDE-DNA测量进行调整，并将具有使用手术组织进行人类癌症后续研究所需的灵敏度，而不需要大量的DNA。目的一是建立一种用于单分子检测BPDE-DNA加合物的固态纳米孔测量系统。目标2将开发一种方案，用于对暴露于BaP或BPDE的细胞系的DNA中的BPDE加合物进行基因组图谱绘制。这项研究将提供“概念证明”和科学证据，据此可以设计后续实验，评估特定人群中的苯并（a）芘暴露情况。实现这些目标将开辟新的研究领域，并提供有价值的新工具，快速和详细的测量长期和短期接触的苯并（a）芘。这里介绍的方法可以扩展到大多数其他毒素和致癌物质，形成DNA加合物。这些发现将有利于癌症和科学研究的各个领域，例如研究加合物的毒理学。