Bentolila Laurent A
University of Chemistry and Biochemistry, California NanoSystems Institute, University of California at Los Angeles, Los Angeles, CA, USA.
Methods Mol Biol. 2010;659:147-63. doi: 10.1007/978-1-60761-789-1_10.
Coming from the material sciences, fluorescent semiconductor nanocrystals, also known as quantum dots (QDs), have emerged as powerful fluorescent probes for a wide range of biological imaging applications. QDs have several advantages over organic dyes which include higher brightness, better resistance to photobleaching, and simplified multicolor target detection. In this chapter, we describe a rapid assay for the direct imaging of multiple repetitive subnuclear genetic sequences using QD-based FISH probes. Streptavidin-coated QDs (SAvQDs) are functionalized with short biotinylated oligonucleotides and used in a single hybridization/detection step. These QD-FISH probes penetrate both intact interphase nuclei and metaphase chromosomes and show good targeting of dense chromatin domains. Importantly, the broad absorption spectra of QDs allows two sequence specific QD-FISH probes of different colors to be simultaneously imaged with a single laser excitation wavelength. This method, which requires minimal custom conjugation, is easily expandable and offers the experimentalist a new alternative to increase flexibility in multicolor cytogenetic FISH applications of repetitive DNAs.
源自材料科学的荧光半导体纳米晶体，也被称为量子点（QDs），已成为用于广泛生物成像应用的强大荧光探针。与有机染料相比，量子点具有几个优点，包括更高的亮度、更好的抗光漂白性以及简化的多色目标检测。在本章中，我们描述了一种使用基于量子点的荧光原位杂交（FISH）探针直接成像多个重复亚核基因序列的快速检测方法。链霉亲和素包被的量子点（SAvQDs）用短的生物素化寡核苷酸进行功能化，并用于单个杂交/检测步骤。这些量子点-FISH探针可穿透完整的间期细胞核和中期染色体，并显示出对致密染色质结构域的良好靶向性。重要的是，量子点的宽吸收光谱允许两种不同颜色的序列特异性量子点-FISH探针在单一激光激发波长下同时成像。这种方法所需的定制偶联最少，易于扩展，并为实验人员提供了一种新的选择，以增加在重复DNA的多色细胞遗传学FISH应用中的灵活性。
