Manipal Institute of Regenerative Medicine, Manipal Academy of Higher Education, Yelahanka, Bengaluru, India.
Bengaluru, India.
Curr Protoc. 2023 May;3(5):e785. doi: 10.1002/cpz1.785.
The analysis of chromosomes by flow cytometry is termed flow cytogenetics, and it involves the analysis and sorting of single mitotic chromosomes in suspension. The study of flow karyograms provides insight into chromosome number and structure to provide information on chromosomal DNA content and can enable the detection of deletions, translocations, or any forms of aneuploidy. Beyond its clinical applications, flow cytogenetics greatly contributed to the Human Genome Project through the ability to sort pure populations of chromosomes for gene mapping, cloning, and the construction of DNA libraries. Maximizing the potential of these important applications of flow cytogenetics relies on precise instrument setup and optimal sample processing, both of which impact the accuracy and quality of the data that are generated. This article is a compilation of the existing protocols that describe the stepwise methodology of accumulating, isolating, and staining metaphase chromosomes to prepare single-chromosome suspensions for flow cytometric analysis and sorting. Although the chromosome preparation protocols have remained largely unchanged, cytometer technology has advanced dramatically since these protocols were originally developed. Advances in cytometry technologies offer new and exciting approaches for understanding and monitoring chromosomal aberrations, but the hallmark of these protocols remains their simplicity in methodologies and reagent requirements and the accuracy of data resolvable to every chromosome of the cell. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mitotic block and cell harvesting Basic Protocol 2: Propidium iodide isolation Support Protocol 1: Swelling test Basic Protocol 3: MgSO low-molecular-weight isolation Basic Protocol 4: Polyamine high-molecular-weight isolation Support Protocol 2: Molecular-weight determination of chromosomal DNA Basic Protocol 5: Chromosome analysis and sorting.
通过流式细胞术对染色体进行分析称为流式细胞遗传学，它涉及悬浮液中单丝分裂染色体的分析和分选。对流动核型图的研究提供了对染色体数量和结构的深入了解，提供了有关染色体 DNA 含量的信息，并能够检测缺失、易位或任何形式的非整倍体。除了其临床应用外，流式细胞遗传学还通过能够对纯染色体群体进行分类以进行基因定位、克隆和 DNA 文库构建，为人类基因组计划做出了巨大贡献。最大限度地发挥流式细胞遗传学的这些重要应用潜力取决于精确的仪器设置和最佳的样品处理，这两者都影响生成数据的准确性和质量。本文汇集了现有的方案，这些方案描述了积累、分离和染色中期染色体的逐步方法，以制备用于流式细胞分析和分选的单个染色体悬浮液。尽管染色体制备方案在很大程度上保持不变，但自这些方案最初开发以来，细胞仪技术已经取得了巨大的进步。细胞术技术的进步为理解和监测染色体异常提供了新的令人兴奋的方法，但这些方案的标志仍然是其方法和试剂要求的简单性以及可分辨每个细胞染色体的数据的准确性。© 2023 作者。Wiley 期刊出版的当前方案。基本方案 1：有丝分裂阻断和细胞收获基本方案 2：碘化丙啶分离支持方案 1：膨胀试验基本方案 3：MgSO 低分子量分离基本方案 4：多胺高分子量分离支持方案 2：染色体 DNA 分子量的测定基本方案 5：染色体分析和分选。
