A novel single-molecule telomere characterization technology for analyzing cancer

用于分析癌症的新型单分子端粒表征技术

• 批准号: 8664139
• 负责人: Harold RIETHMAN
• 金额: $ 30.46万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-13 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8664139
• 关键词: Alleles; Apoptosis; Bar Codes; Base Pairing; Biological; Biological Assay; Biology; Cancer cell line; Cells; Chromosomes; Collection; DNA; Data; Detection; Development; Diploidy; Event; Fibroblasts; Fluorescent Dyes; Functional disorder; Genome; Genomic DNA; Genomic Instability; Genomics; Goals; Human; Image; Individual; Interphase Cell; Label; Length; Link; Maintenance; Malignant Neoplasms; Measurement; Measures; Mediating; Methods; Miniaturization; Molecular; Mutation; Neoplasm Circulating Cells; Pattern; Performance; Play; Population; Process; Regulation; Resolution; Role; Sampling; Scheme; Signal Transduction; Site; Sorting - Cell Movement; Specificity; Staging; Stratification; Stretching; System; Technology; Telomerase; Testing; Time; anticancer research; base; cancer cell; cancer genome; cancer stem cell; carcinogenesis; design; ds-DNA; high throughput analysis; insight; nanochannel; neoplastic cell; novel; prognostic; public health relevance; research study; senescence; single molecule; technology development; telomere; tool; tumor

DESCRIPTION (provided by applicant): Single-telomere-specific (TTAGGG)n tract lengths and instabilities cannot be measured globally using current methods; the ability to do so would be a major, transformative addition to the arsenal of tools for analyzing critical telomere loss an telomere elongation events in cancer. The shortest telomere or a small subset of the shortest telomeres in a cell will determine the onset of senescence, apoptosis, or genome instability; single (TTAGGG)n tracts are crucial for the function of telomeres and the biological effects of telomere attrition and dysfunction. Telomere loss, breakage, fusion, and rejoining are highly elevated in cancer, but current methods for detecting and measuring these mutational events at the molecular level are limited and low-throughput. The technology we propose to develop here would permit quantitative, single- allele-resolution measurements of telomere length and instability, enabling unprecedented insights into the role(s) telomere loss, telomere breakage/re-joining, and telomerase or ALT dependent telomere elongation play in carcinogenesis, including mechanistic insights into molecular events mediating these processes and translational insights for the potential prognostic and tumor stratification applicability of the methods. In this method, input genomic DNA is labeled with fluorescent dyes specific for (TTAGGG)n sequences and for linked subtelomeric DNA. The labeled individual DNA fragments are linearized (stretched) and imaged in the nano-channels of Bionano Genomics system at very high throughput. The lengths of the telomere are measured accurately and the distances between probes in the subtelomere region are determined accurately to infer the identity of the telomere. Our goals for this R21 study are to establish feasibility for (1) high-throughput single-molecule detection and quantitation of (TTAGGG)n tracts in genomic DNA samples and (2) subtelomere probe development and efficient co-labeling of telomeres and subtelomeres in the context of total genomic DNA, including conversion of the labeled DNA to double- stranded DNA suitable for nano-channel analysis; and (3) proof-of-principle results for the technology and its applicability for cancer research using data generated and analyzed for a normal and a cancer cell line. There are significant technical challenges inherent in this early-stage technology development project, but the extraordinary payoff for cancer research will be a high-throughput ability to probe mechanisms of telomere length regulation and telomere mutation at single-telomere resolution, in small (ultimately single-cell) samples of both dividing and non-dividing cells.

描述（由申请人提供）：单层特异性（TTAGGG）N区域的长度和不稳定性无法使用当前方法在全球范围内测量；这样做的能力将是用于分析癌症中端粒伸长事件的工具武器库的主要，变革性的补充。细胞中最短的端粒或最短端粒的一小部分将决定衰老，凋亡或基因组不稳定性的发作。单个（Ttaggg）N区对于端粒功能以及端粒损耗和功能障碍的生物学作用至关重要。癌症中的端粒损失，断裂，融合和重新加入癌症高度升高，但是当前检测和测量这些突变事件在分子水平上的方法是有限的，并且低通量。我们建议在这里开发的技术将允许对端粒长度和不稳定的定量，单等位基因分辨率测量，从而使端粒损失的前所未有的见解，端粒损坏，断裂/重新加入/重新加入/端粒酶，端粒酶或Alt依赖性端粒息肉在包括机械化的这些范围的范围内，包括机械化的事件，使这些型号的变化构成了这些固有的插入式插入式的插入式插入式插入式插入式插入式插入，以使这些机械插入式介绍到固有的近端，以使这些型号的插入型插入式介绍，以使这些端流变为固有的物质插入式，以构成的机械性插入式介绍了分解的分类属性。方法的预后和肿瘤分层适用性。在这种方法中， 输入基因组DNA用特异性（TTAGGG）N序列和链接的亚电体DNA的荧光染料标记。标记的单个DNA片段是线性化的（拉伸），并在Bionano基因组系统的纳米通道中成像，并以非常高的吞吐量成像。准确测量端粒的长度，并准确地确定亚电球区域的探针之间的距离，以推断端粒的身份。我们的这项R21研究的目标是确定（1）基因组DNA样本中（TTAGGG）n段的高通量单分子检测和定量（2）（2）（2）亚电位探针开发以及有效的共同标记的端粒和端粒和亚电位的共同标记，包括dna dna dna，包括dna dna dna dna dna dna dna dna dna dna dna dna dna dna的范围。纳米通道分析； （3）使用对正常和癌细胞系生成和分析的数据，该技术的原则结果及其在癌症研究中的适用性。这个早期技术开发项目固有存在重大的技术挑战，但是癌症研究的非凡收益将是高通量能力，可以在单telomere分辨率下探测端粒长度调节和端粒突变的机制，在小（最终是单细胞）样品中的分裂和非分裂细胞的样品中。