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Publisher Summary This chapter presents chromosome sorting by flow cytometry. Flow cytometers operate on the principle of rapid analysis of single cells or chromosomes. A suspension of fluorescently stained chromosomes flows at a rate between 1000 to 2000/sec through a finely focused laser beam. Individual chromosome types are resolved on the basis of DNA content and base composition as determined by the cytochemistry of the fluorescent dyes. The chromosomes flow in a small stream that is broken into uniform droplets. The droplet containing the desired chromosome type, as indicated by the fluorescence measurement, can be deflected from the mainstream. In addition to the operation of the flow cytometer, the culturing of cells and chromosome preparation techniques must be highly efficient. A chromosome preparation should provide uniformly stained chromosomes, low debris levels, a high yield of free chromosomes from mitotic cells, and for library construction, chromosomal DNA of very high molecular weight. The yield of free chromosomes is crucial because low yields decrease the chromosome number concentration while increasing the debris fraction, making the chromosome sorting rates much slower than optimum. Techniques for chromosome preparation, resolution of the normal human chromosomes for flow sorting, and the way these interact to produce sorted chromosomes of high purity at optimum rates are described in the chapter. |
