Fluorescence in situ Hybridization (FISH).
Fluorescence in situ Hybridization (FISH).
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DOI：
10.1002/0471143030.cb2204s23
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发表时间：
2004-09-01
影响因子：
--
通讯作者：
Squire, Jeremy A
中科院分区：
文献类型：
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作者：
Bayani, Jane;Squire, Jeremy A
荧光原位杂交（FISH）涉及两个主要组分的制备：DNA探针和探针将与之杂交的靶DNA。DNA探针通常来自克隆的来源，例如质粒、质粒、PAC、YAC或BAC;其中插入物可以含有特定基因或源自特定染色体基因座。也可以使用全染色体涂料，但通常适用于中期制备。然后可以使用半抗原间接标记和检测纯化的DNA，或者使用荧光染料或染料缀合的核苷酸直接标记。标记策略也是可变的，采用标准切口平移或PCR标记方法。靶DNA可以采取染色体扩散或间期核的形式。间期靶点的来源可能来自细胞遗传学制备物或石蜡包埋组织。标记的DNA探针和DNA靶均变性为单链状态并允许彼此杂交。在24小时杂交之后进行杂交后洗涤和荧光标记抗体孵育，并且样品准备好用于通过荧光显微镜观察。FISH实验的成功解释取决于起始材料的质量、杂交效率以及杂交后洗涤和抗体检测的严格性。
Fluorescence in situ Hybridization (FISH) involves the preparation of two main components: the DNA probe and the target DNA to which the probe will be hybridized. The DNA probe typically comes from cloned sources such as plasmids, cosmids, PACs, YACs, or BACs; where the insert may contain a specific gene or originate from a specific chromosomal locus. Whole-chromosome paints may also be used but are usually applicable to metaphase preparations. The purified DNA can then be labeled and detected indirectly using haptens, or labeled directly using fluorochrome or dye-conjugated nucleotides. Labeling strategies are also variable, employing standard nick translation or PCR labeling methods. The target DNA can take the form of chromosomes spreads or interphase nuclei. The sources of interphase targets may come from cytogenetic preparations or from paraffin-embedded tissues. Both the labeled DNA probe and DNA target are denatured to a single-stranded state and permitted to hybridize to each other. Post-hybridization washes and fluorescently-labeled antibody incubations follow the 24-hour hybridization, and the specimen is ready for visualization by fluorescent microscopy. Successful interpretation of FISH experiments is dependent on the quality of the starting materials, hybridization efficiencies, and stringency of post-hybridization washes and antibody detections.