Nguyen Ha Nam, Reijo Pera Renee A
Center for Human Embryonic Stem Cell Research and Education, Institute for Stem Cell Biology and Regenerative Medicine, Department of Obstetrics and Gynecology, Stanford University, Palo Alto, CA 94304-5542, USA.
CSH Protoc. 2008 Sep 1;2008:pdb.prot5047. doi: 10.1101/pdb.prot5047.
INTRODUCTIONSpectral karyotyping is a powerful technique for enumeration of chromosomes in plant, animal, and human cells with high precision. It is also used as a tool for detecting and diagnosing abnormalities in individual chromosomes, such as deletions, duplications, and translocations. There are three basic steps to obtaining good spectral karyotypes: (1) making metaphase spreads, (2) hybridizing probes to the DNA, and (3) analyzing the spectral characteristics of the chromosomes. There are two techniques for spectral karyotyping that are widely used: spectral karyotype (SKY) and multicolor fluorescence in situ hybridization (M-FISH). Both techniques use multicolored fluorescent probes which emit at different wavelengths (200-700 nm) upon light excitation to hybridize to the target DNA sequences. The methods differ in the way in which light is captured and analyzed by the corresponding software. In this protocol, methods for making metaphase spreads from human embryonic stem cells (hESCs) for spectral karyotyping (SKY) using the spectral imaging system and software from Applied Spectral Imaging are described.
引言
光谱核型分析是一种用于精确计数植物、动物和人类细胞中染色体的强大技术。它还被用作检测和诊断单个染色体异常的工具，如缺失、重复和易位。获得良好光谱核型有三个基本步骤：（1）制备中期染色体铺片，（2）将探针与DNA杂交，（3）分析染色体的光谱特征。有两种广泛使用的光谱核型分析技术：光谱核型（SKY）和多色荧光原位杂交（M-FISH）。这两种技术都使用多色荧光探针，在光激发下会在不同波长（200-700nm）发射荧光，以与目标DNA序列杂交。这两种方法在相应软件捕获和分析光的方式上有所不同。在本方案中，将描述使用应用光谱成像公司的光谱成像系统和软件从人类胚胎干细胞（hESC）制备用于光谱核型分析（SKY）的中期染色体铺片的方法。
