Introduction to the Workshop
URLs Provided by Attendees

Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

Infrastructure

The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

TIME-GATED FLOW ANALYSIS OF HUMAN CHROMOSOMES

V.V. Zenin#, N.D. Aksenov#, A.N. Shatrova#, Y. V. Kravatsky*, A. Kuznetsova*, L. S. Cram^, A. I. Poletaev*;
#Institute of Cytology, RAS, St. Petersburg; *Engelhardt Institute of Molecular Biology, RAS, Moscow; ^Los Alamos National Laboratory, Los Alamos, NM 87545.

For chromosome analysis and sorting we have made several modifications to our flow cytometer and chromosome isolation procedures. An electronics modification of our serial flow-sorter (ATC-3000, ODAM-Bruker) was made to provide the option of measuring the inter-event time interval in the microsecond to millisecond time range. The inter-event time interval can be used as an independent parameter in flow cytometry data acquisition. A stepper motor driven sample delivery system (for achieving calibrated flow rates) is used in conjunction with a capillary high-gradient mitotic cell breaking device to disrupt mitotic cells and release intact metaphase chromosomes. We have found that one can get stable staining of human chromosomes within mitotic cells with both Hoechst 33258 and Chromomycin A3 without significant cell membrane rupture using saponin as the cell membrane affecting substance. Two versions of a mitotic cell breaking device were tested: electromagnet shearing and a high-gradient curved capillary. Both devices proved to be compatible with most of serial flow cytometers able to perform flow analysis of chromosomes. Time-gated data acquisition provides a possibility to filter out signals of chromosomes arising from single cells from contaminating signals: cell debris and randomly appearing single chromosomes. This can be done both in real time and on list mode data files. The specific software provides the possibility of doing different filtering procedures with list mode data to get the differential information about intracellular chromosome sets. Future work will target optimization of the new components and the procedure of measurement. The realization of this approach might provide an efficient analysis of different chromosome aberrations. This work was supported by grants from DOE and Russian State Program "Human Genome".