Research Project
3443886
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.
