OSEEN FORCE CYTOMETRY/SORTING

每个人类基因组中心被分配来绘制或排序特定的 染色体将从稳定的染色体供应中受益。大型 需要大量的纯染色体才能产生特定的文库和 目的：利用原位探针杂交技术进行基因定位(1)。目前， 用流式细胞仪对染色体进行分类。最快的分拣速度 用这项技术实现的给定染色体的频率是几百赫兹， 它可以产生足够的特定DNA来构建噬菌体文库 下午(2)。作为粘粒中较大片段的染色体文库(3)和 酵母人工染色体(4，5)将需要10倍或100倍的 特定的DNA。 我们建议满足人类基因组计划对纯化的需求 通过开发分离染色体的仪器来分离染色体 在声波场中使用声悬浮。由此产生的奥西力 基波与其二次谐波的相互作用(6) 将被用来在气相中将能量传递给分离的染色体。 声波随垂直距离的衰减产生一个梯度 在与恒定重力相反的奥西力中。一条染色体迁移到 作为大小和形状的函数的平衡位置。有几个 与流分类方法相比，这种方法具有以下优势： 1)一次对所有染色体进行排序。 2)染色体在物理上与碎片分开。 3)该设备设计相对简单，易于扩展到更高 样本吞吐量。其中许多都可以以适中的成本建造。 4)分离后的染色体可以集中到较高浓度。 5)不需要进行荧光DNA染色。 除了分拣仪器的开发外，可靠的分拣方法 将制定用于排序的染色体准备。的发展。 可分离大分子DNA的完整染色体制备方法 并回收用于分子生物学实验是一个有价值的目标 就凭它自己的力量。尤其是如果染色体上没有 溶剂稳定，可进行非水相和气相操纵。分析性 将建立程序来评估回收的DNA的完整性 使用所提出的方法。

Each Human Genome Center assigned to map or sequence a particular chromosome would benefit from a steady supply of that chromosome. Large numbers of pure chromosomes are required to generate specific libraries and to facilitate gene mapping by in situ probe hybridization (1). At present, chromosomes are sorted with flow cytometers. The fastest sorting rate achieved with this technology is several hundred Hz for a given chromosome, which can generate enough specific DNA for a bacteriophage library in an afternoon (2). Chromosome libraries as larger segments in cosmids (3) and yeast artificial chromosomes (4,5) will require 10 or 100 times as much specific DNA. We propose to address the need of the Human Genome Program for purified chromosomes by developing instrumentation that fractionates chromosomes using acoustic levitation in a sound wave field. The Oseen force resulting from interaction of a fundamental sound wave with its second harmonic (6) will be used to transmit energy to isolated chromosomes in the vapor phase. Attenuation of the acoustic wave with vertical distance creates a gradient in the Oseen force opposed by constant gravity. A chromosome migrates to an equilibrium position as a function of size and shape. There are several advantages to such an approach in comparison with flow sorting methods: 1)all of the chromosomes are sorted at once. 2)chromosomes are physically segregated from debris. 3)the device is relatively simple in design and readily scaleable to higher sample throughputs. Many could be built at modest cost. 4)fractionated chromosomes can be focused to high concentrations. 5)fluorescent DNA stains are not required. In addition to the development of a sorting instrument, reliable methods of preparing chromosomes for sorting will be formulated. The development of methods to prepare whole chromosomes from which large DNA can be separated and recovered for molecular biological experimentation is a valuable goal in its own right. This is especially true if the chromosomes are free of solvent and stable to non-aqueous and vapor phase manipulation. Analytical procedures will be established to evaluate the integrity of DNA recovered using the proposed methods.