Dobrinski Kimberly P, Brown Kim H, Freeman Jennifer L, Lee Charles
Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, USA; Harvard Medical School, Boston, Massachusetts, USA.
Methods Cell Biol. 2011;104:237-57. doi: 10.1016/B978-0-12-374814-0.00014-8.
Molecular cytogenetics is a field that emerged in the 1980s, based on a technique referred to as fluorescence in situ hybridization, (FISH). Using FISH methodologies, a specific DNA sequence or collection of DNA fragments may be selectively labeled with a hapten molecule or fluorescent dye and hybridized to denatured chromosomes, interphase cells, or even chromatin fibers. DNA hybridization kinetics permit these labeled probes to anneal to their complementary sequences on such chromosomal DNA preparations allowing for direct visualization of the sequence of interest in the genome being interrogated. If present, the relative chromosomal position of the sequence can sometimes also be ascertained. Progress in molecular cytogenetic research has advanced the genetic characterization of zebrafish models of human diseases as well as assisted with accurate annotation of the zebrafish reference genome by anchoring large DNA fragments to specific chromosome regions. Using the procedures described in this chapter, hundreds of ambiguous zebrafish bacterial artificial chromosome (BAC) clones have already been assigned to individual genetic linkage groups. Molecular cytogenetic techniques can also be used to study gene duplication events and study the molecular mechanisms by which they arise. Moreover, the availability of a new molecular cytogenetic technique, array-based comparative genomic hybridization (aCGH), is now able to identify gains and losses of DNA segments in zebrafish DNA samples in a genome-wide manner and in a single assay.
分子细胞遗传学是一个在20世纪80年代兴起的领域，它基于一种被称为荧光原位杂交（FISH）的技术。使用FISH方法，特定的DNA序列或DNA片段集合可以用半抗原分子或荧光染料进行选择性标记，并与变性的染色体、间期细胞甚至染色质纤维杂交。DNA杂交动力学允许这些标记的探针与其在这类染色体DNA制剂上的互补序列退火，从而可以直接观察被检测基因组中感兴趣的序列。如果存在，有时还可以确定该序列在染色体上的相对位置。分子细胞遗传学研究的进展推动了人类疾病斑马鱼模型的遗传特征分析，同时通过将大的DNA片段锚定到特定染色体区域，辅助了斑马鱼参考基因组的精确注释。使用本章所述的方法，数百个模糊的斑马鱼细菌人工染色体（BAC）克隆已经被分配到各个遗传连锁群。分子细胞遗传学技术还可用于研究基因复制事件及其发生的分子机制。此外，一种新的分子细胞遗传学技术——基于微阵列的比较基因组杂交（aCGH），现在能够在全基因组范围内并通过一次检测识别斑马鱼DNA样本中DNA片段的增减情况。
