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Hybrid approach for comprehensive mutation detection in a cell

用于细胞内全面突变检测的混合方法

基本信息

批准号：
10662613
负责人：
ALEXEJ ABYZOV
金额：
$ 36.57万
依托单位：
MAYO CLINIC ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-15 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10662613
关键词：
Address Anatomy Autopsy Benchmarking Biopsy Bladder Bone Marrow Cells Cell Line Cell Proliferation Cells Characteristics Chondrocytes Clonal Expansion Clone Cells Cloning Colon Complex Cornea Cryopreserved Cell Cytogenetics DNA DNA amplification DNA biosynthesis Development Disadvantaged Endothelial Cells Epithelium Equilibrium Esophagus Fibroblasts Freezing Frequencies Genetic Genome Hair follicle structure Haplotypes Heart Heterogeneity Human Human body Hybrids In Vitro Intestines Joints Kidney Limb structure Lung Methods Morphologic artifacts Mosaicism Muscle Cells Mutation Mutation Detection Nature Organ Pathway Analysis Phase Polymerase Proliferating Protocols documentation Repetitive Sequence Scalp structure Skin Skin Tissue Somatic Mutation Stomach System Testing Tissue Preservation Tissues Work biobank cell type genome analysis human tissue melanocyte mosaic novel strategies preservation proliferation potential single cell analysis stem cells success whole genome zygote

项目摘要

Abstract Numerous recent studies have consistently shown that likely no two cells in the human body have the same genomes, a phenomenon called somatic mosaicism. Mosaicism can be studied using various approaches, but the study of mutations directly in the cell promises a comprehensive characterization of mosaicism in any tissue. Analysis of single cell genome by cloning relies on natural DNA replication machinery in cells and, thus, minimizes errors in DNA during cloning; however, cloning is limited by the ability of cells to proliferate. Analysis by whole genome amplification (WGA) is hampered by introduced errors and non-uniformity of amplification. Here we propose to address the limitations of single cell cloning and single cell WGA by developing a hybrid approach that proceeds in two stages: 1) limited culturing of single cells to a micro-sized colony of 2-50 cells; and 2) WGA of the micro-size colonies to yield enough DNA material for sequencing. An optimized hybrid approach will enable rigorously and unbiasedly studying somatic mosaic at a single cell level throughout the human body without WGA artifacts. Finally, to preserve tissue cell heterogeneity and enable biobanking of tissues amenable to the developed hybrid approach, we will develop a storing protocol for tissues to preserve proliferative potential of cells in the stored tissues. Success of the project would enable comprehensive and accurate discovery of mutations in a single cell in a variety of tissues prioritized by SMaHT and beyond, deepening our understanding of the mosaicism of humans. 2

摘要