Development of new tools for Single Cell Analysis

开发单细胞分析新工具

• 批准号: 8423040
• 负责人: DAVID R. WALT
• 金额: $ 32.34万
• 依托单位: TUFTS UNIVERSITY MEDFORD
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-11-18 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8423040
• 关键词: Address; Age; Age Factors; Aging; Alleles; Area; Bacterial Antibiotic Resistance; Bacterial Infections; Behavior; Biochemistry; Biological Assay; Biology; Biomedical Engineering; Cells; Cellular biology; Chemistry; Chromosome Mapping; Clinical; Complex; Cytolysis; DNA; Data Set; Detection; Development; Diagnostic; Disease; Enzymes; Escherichia coli; Exhibits; Fiber Optics; Frequencies; Genetic; Genetic Crosses; Genetic Population Study; Genetic Recombination; Genetic Variation; Genome; Genomics; Genotype; Goals; Heterogeneity; Individual; Investigational Therapies; Lead; Malignant Neoplasms; Maps; Masks; Measurement; Messenger RNA; Methods; Modeling; Molecular Biology; Molecular Structure; Monitor; Neoplasm Metastasis; Normal Cell; Outcome; Phenotype; Population; Population Genetics; Principal Investigator; Proteins; Regimen; Regulator Genes; Relative (related person); Research Personnel; Resolution; SNP genotyping; Sampling; Somatic Mutation; Surface; System; Technology; Therapeutic; Tissues; Variant; base; biological research; cell type; cost; graduate student; human male; improved; interest; neoplastic cell; new technology; novel; public health relevance; response; single cell analysis; single molecule; sperm cell; statistics; success; tool; tumor progression

DESCRIPTION (provided by applicant): Single cell analysis is becoming increasingly important as it is clear that ensemble measurements mask the diversity of the biology in cell populations. Single cell genotyping and phenotyping are necessary to define functional heterogeneity in varying cell types including diseased vs. normal cells. The overall goal of this project is to develop new bioanalytical tools for performing high resolution single cell analysis. This interdisciplinary project involves Chemistry, Molecular Biology, Biochemistry, Genetics, and Bioengineering and will be conducted by a team including a senior Principal Investigator, one postdoctoral associate, and two graduate students. Specific Aim 1-Develop a high fidelity single DNA molecule genotyping approach for analyzing thousands of molecules simultaneously and with the ability to interrogate multiple SNPs. We plan to employ fiber optic microwell arrays to develop a robust, efficient, fast, and affordable method of simultaneously genotyping single DNA molecules from thousands of individual cells. Specific Aim 2-Develop an approach to isolating single cells, capturing genomic DNA, and genotyping the captured DNA. In this Aim, we will apply the methods developed in Specific Aim 1 to whole cells. Thousands of single E. coli cells will be captured in individual wells, lysed, genomic DNA fragmented, the DNA captured on the surface of the individual wells, and then genotyped. Specific Aim 3-Develop a high resolution single molecule analysis method for analyzing the contents of single cells. We will develop methods for performing high resolution single molecule counting of the contents of individual cells. Single cells will be isolated, lysed, and their contents will be captured in microwell arrays such that individual mRNA and protein molecules are isolated. We will then use a variety of methods that enable us to count the individual molecules. Success of these aims will provide a powerful new technology broadly applicable to multiple areas of biological research. For biologists, it provides the ability to describe the complex genetics of cell populations, providing clinicians with improved opportunities for diagnostics, and relating population genetics with responses to therapy and clinical outcomes. For experimentalists, it provides new ways to study phenomena such as tumor progression and responses to experimental therapies. For researchers on aging, it provides the ability to monitor changes in somatic genetics with age and the factors influencing them. For geneticists, it provides a low cost ability to construct genetic maps, to identify trans-acting elements controlling recombination, and to monitor the molecular structure of recombination products with high resolution. Our long term goal is to apply the developed technology to a variety of cell types to study fundamental cell biology and to understand how cell-to-cell and cell population differences may lead to different cell fates and may define disease.

描述（由申请人提供）：单细胞分析变得越来越重要，因为很明显，合奏测量掩盖了细胞种群中生物学的多样性。单细胞基因分型和表型对于在包括患病与正常细胞在内的不同细胞类型中定义功能异质性是必要的。该项目的总体目标是开发用于执行高分辨率单细胞分析的新生物分析工具。这个跨学科项目涉及化学，分子生物学，生物化学，遗传学和生物工程，并将由一个团队进行，包括高级首席研究员，一名博士后同学和两名研究生。特定的目标1开发一种高保真度单DNA分子基因分型方法，用于同时分析数千个分子并具有询问多个SNP的能力。我们计划采用光纤微孔阵列来开发一种稳健，有效，快速且负担得起的方法，用于从数千个单个细胞中同时基因分型单型单个DNA分子。特定的目标2开发方法是隔离单细胞，捕获基因组DNA和基因分型捕获的DNA的方法。在此目标中，我们将应用特定目标1中开发的方法对整个细胞。成千上万的单一大肠杆菌细胞将被捕获在单个井中，裂解，基因组DNA碎片，在单个井表面捕获的DNA，然后进行基因分型。特定目标3开发一种高分辨率的单分子分析方法，用于分析单细胞的含量。我们将开发用于执行单个细胞含量的高分辨率单分子计数的方法。单个细胞将被分离，裂解，其含量将在微孔阵列中捕获，从而分离单个mRNA和蛋白质分子。然后，我们将使用各种方法，使我们能够计算单个分子。这些目标的成功将提供一项强大的新技术，广泛适用于生物学研究的多个领域。对于生物学家，它提供了描述细胞群体复杂遗传学的能力，为临床医生提供了改善诊断机会，并将人群遗传学与对治疗和临床结果的反应有关。对于实验者来说，它提供了研究现象的新方法，例如肿瘤进展和对实验疗法的反应。对于衰老研究人员，它提供了随着年龄的增长以及影响它们的因素来监测体细胞遗传学变化的能力。对于遗传学家来说，它提供了构建遗传图，识别控制重组的跨作用元件以及以高分辨率监测重组产物的分子结构的低成本能力。我们的长期目标是将开发的技术应用于各种细胞类型，以研究基本细胞生物学，并了解细胞对细胞和细胞群体的差异如何导致不同的细胞命运并可能定义疾病。

项目成果

Assessing the stochastic intermittency of single quantum dot luminescence for robust quantification of biomolecules.

• DOI: 10.1021/ac4001332
• 发表时间: 2013-07-16
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Palacios, Manuel A.;Lacy, Michael M.;Schubert, Stephanie M.;Manesse, Mael;Walt, David R.
• 通讯作者: Walt, David R.