OSEEN FORCE CYTOMETRY/SORTING

每个人类基因组中心都被分配到一个特定的基因组， 染色体将受益于该染色体的稳定供应。 大 需要大量的纯染色体来产生特定的文库， 以便于通过原位探针杂交进行基因定位（1）。 目前， 用流式细胞仪分选染色体。 最快的分拣速度 对于给定的染色体，该技术可以实现数百赫兹的频率， 它可以为噬菌体文库产生足够的特异性DNA， 下午（2） 染色体文库作为Cosmos中的较大片段（3）和 酵母人工染色体（4，5）将需要10或100倍 特定的DNA 我们建议解决人类基因组计划的需要， 通过开发分离染色体的仪器 使用声波场中的声悬浮。 Oseen力导致 从基本声波与其二次谐波（6）的相互作用 将被用来传输能量到气相中的分离染色体。 声波随垂直距离的衰减产生梯度 与恒重力对抗的奥森力 一个染色体迁移到 作为大小和形状的函数的平衡位置。 有几 与流分类方法相比，这种方法的优点是： 1)所有的染色体同时被分类。 2)染色体与碎片物理分离。 3)该装置在设计上相对简单 样品吞吐量。 许多可以以适度的成本建造。 4)分级分离的染色体可以被集中到高浓度。 5)不需要荧光DNA染色剂。 除了开发分选仪器外，还需要可靠的分选方法。 准备用于分选的染色体将被制定。 的发展 制备可分离大DNA的完整染色体的方法 并回收用于分子生物学实验是一个有价值的目标 在它自己的权利。 如果染色体不含 溶剂且对非水相和气相操作稳定。 分析 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.