Hutchison N J, Langer-Safer P R, Ward D C, Hamkalo B A
J Cell Biol. 1982 Nov;95(2 Pt 1):609-18. doi: 10.1083/jcb.95.2.609.
In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.
原位杂交已成为在细胞学标本中定位DNA或RNA序列的标准方法。我们开发了两种方法，以小鼠卫星DNA与整装中期染色体杂交作为测试系统，将该技术扩展到透射电子显微镜水平。设计的第一种方法是标准光学显微镜水平的直接延伸，以小鼠卫星DNA与整装中期染色体杂交作为测试系统。设计的第一种方法是标准光学显微镜原位杂交的直接延伸。放射性标记的互补RNA（cRNA）与沉积在电子显微镜网格上并固定在70%乙醇蒸汽中的中期染色体杂交；通过放射自显影检测杂交位点。即使在相对较短的曝光时间后，也能观察到染色体着丝粒区域的特异性和强烈标记。一些中期染色体标本中存在的间期核也显示出卫星DNA标记的明确模式，这表明含卫星的区域在间期相互关联。该方法的灵敏度估计至少与光学显微镜水平相当，而分辨率提高了至少三倍。第二种方法避免了使用放射自显影检测，使用生物素标记的多核苷酸探针。这些探针（DNA或RNA）与网格上固定的染色体杂交后，通过与抗生物素抗体和吸附在胶体金表面的二抗反应来检测杂交体，标记主要集中在着丝粒异染色质及其相关的外周纤维上。标记平均是背景结合的十倍。该方法快速，具有在染色体和染色质中对DNA序列进行精确超微结构定位的潜力。
