14 GATA 8(1): 14-23, 1991

IN SITU HYBRIDIZATION Editor's Note: These articles are the first in a series on in situ hybridization edited by Glen A. Evans. Articles in the next issue of GATA will cover chromosomal in situ hybridization using YACs and localizing DNA and RNA within nuclei and chromosomes.

DNA Sequence Mapping Using Electron Microscopy SANDYA NARAYANSWAMI and BARBARA A. HAMKALO DNA sequences can be mapped on chromosomes at high resolution in the electron microscope after hybridization with a nonisotopically labeled probe followed by detection with a two-step antibody reaction employing a colloidal gold tag. Hybridization probes can be modified with biotin-dUTP. digaxigenin-dll'I'I', dinitrophenyl-dUTP. or Nsacetaxy-l-acetylamlnofluorene (AAF). The availability of different sizes of colloidal gold particles permits the simultaneous detection of several sequences. In addition, low signals can be amplified either with an antibody sandwich scheme or by silver intensification. This technology is applicable both to TEM and SEM preparations of chromosomes. and we have used it to map a number of highly and moderately repeated sequences on whole mount metaphase chromosomes.

Introduction In situ hybridization has become an integral genome mapping technique that allows one to determine the cytological location of cloned sequences of interest. Its use, in conjunction with physical mapping, permits the ordering of linked probes relative to each other and relative to chromosomal structures. The original protocol ernFrom the Department of Molecular Biology and Biochemistry. University of California, Irvine. California. Address correspondence to: Barbara A. Hamkalo, Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717. Received April 20, 1990; revised June 6, 1990; accepted June 25, 1990.

ployed radioactive DNA or RNA probes which, after hybridization to denatured cytological preparations, were detected by autoradiography. This classic procedure has generated a large body of information on the locations of sequences in the chromosomes of a variety of species, from protozoans to humans. Beyond regional localization, Harper and Saunders [11 showed that one could map genes relative to chromosome bands by combining hybridization/detection with standard banding protocols. Despite the utility of this approach, the time required to develop a signal for localization of a low copy sequence and the arduous statistical analysis required to identify a bona fide hybrid site restrict its routine use for rapid localization. The development of nonisotopic labeling regimens in conjunction with detection of specific Ii~ gand interactions by fluorescence or immunoenzymatic assays [2] has placed in situ hybridization in a pivotal position for gene mapping. These labeling regimens are rapid, specific, and efficient. Using these detection systems, numerous single copy sequences have been mapped at the light microscope (LM) level over the past few years. There are a number of reasons for developing equivalent mapping techniques at the electron microscope (EM) level. The greater resolution of the EM presents the opportunity to determine relative map positions of sequences that cannot be discriminated readily in the LM. It allows high resolution mapping within chromosomal landmarks such as centromeres, telomeres, and secondary constrictions. In addition, it is possible to analyze the sequence organization of small chromosomes such as double-minutes and small marker chromosomes. Finally, one can map sequences in small nuclei such as yeast, in order to exploit the most well characterized eukaryotic genetic system for studies on nuclear structure and function. Chromosome mapping in the EM was developed by Hutchison et al. [3] by combining hybridization of biotin-labeled probes to whole-mount mitotic chromosomes on EM grids with a twostep antibody reaction and colloidal gold detection of hybrid sites. Conoidal gold is the detection system of choice because of the pronounced electron density of gold particles, their low background binding to chromosomes, and the availability of particles in different sizes allowing simultaneous detection of multiple probes. The original protocol has been modified and improved

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15 DNA Sequence Mapping

GATA 8(1): 14-23, 1991

to increase signal intensity and reduce background [4]. The modified technique has been used to map centromeric and telomeric sequences in the mouse, a number of moderately repetitive coding sequences in Xenopus, and a variety of other highly and moderately repeated sequences in vertebrate chromosomes [5-8] (Narayanswami et aI., manuscript in preparation). This paper describes the methodology currently employed and some of the past and possible future applications of this technique.

Methodology A schematic representation of the basic protocol is presented in Figure 1. Figure 1. A schematic representation of th~ procedure followed for EM in situ hybridization, for both single and double labeling .

Cytological Preparations Hybridization is carried out on parlodion-carboncoated gold EM grids to which whole mount metaphase chromosomes are bound. Cultured cells are arrested in metaphase by either colcemid or nocodazole, and the mitotic cells are collected by shake-off, lysed in a 1% solution of Nonidet P-40, and centrifuged onto EM grids through a cushion of I M sucrose essentially as described by Miller et al. [9]. Nuclei can be prepared in the same way, and are often present as low level contaminants in metaphase chromosome preparations. This basic method is applicable to a wide variety of adherent cultured cell lines [5, 7, 8] and takes about 15-30 minutes from the time cells are harvested. It is possible to prepare a large number of grids at one time, which can be stored at room temperature for periods up to at least several months with no obvious reduction in hybridization signal. When suspension cultures are used a

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DNA sequence mapping using electron microscopy.

DNA sequences can be mapped on chromosomes at high resolution in the electron microscope after hybridization with a nonisotopically labeled probe foll...
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