Two techniques are available for high resolution flow cytometry of chromosomes in suspension: Dual Beam Flow Cytometry and SlitScan Flow Cytometry (SSFCM). Dual beam flow cytometry of chromosomes stained with Hoechst 33258 (Ho) and Chromomycin A3 (CA3) and similar dye pairs allows the separation of most but not all human chromosomes. Slitscan flow cytometry and centromeric index (C1) calculation can distinguish between chromosomes that bind equal amounts of Ho and CA3 but differ in the position of the centromere. Thus, chromosomes that are difficult to separate by dual beam flow cytometry (e.g. the larger human chromosomes and especially 9 through 12) can be distinguished by SSFCM. Further, SSFCM allows detection of morphologically aberrant chromosomes such as dicentrics. We propose in this application to develop a flow sorter that allows combined dual beam/slitscan flow cytometric analysis to facilitate classification and purification of the human chromosomes. At the heart of this instrument will be a digital pulse shape analysis circuit that allows C1 calculation or dicentric chromosome detection within 1ms. This circuit and the mechanical and optical components of the dual beam SSFCM will be installed on an existing prototype LLNL sorter thereby augmenting development of a dual beam slitscan sorter (DBSSS). This instrument will be a powerful tool: 1) for the detection of homogeneously occurring numerical and structural aberrations for improved cytogenetic analysis, 2) for quantification of the frequency of random aberrant chromosomes to facilitate quantification of genetic damage, and 3) for purfication of chromosomes according to shape, DNA content, and DNA base composition for validation of the above assays and for improved chromosome purification by sorting.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM025076-09
Application #
3272759
Study Section
(SSS)
Project Start
1978-04-01
Project End
1990-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
9
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Lawrence Livermore National Laboratory
Department
Type
Organized Research Units
DUNS #
827171463
City
Livermore
State
CA
Country
United States
Zip Code
94550
Lucas, J N; Mullikin, J C; Gray, J W (1991) Dicentric chromosome frequency analysis using slit-scan flow cytometry. Cytometry 12:316-22
Lucas, J N; Tenjin, T; Straume, T et al. (1989) Rapid human chromosome aberration analysis using fluorescence in situ hybridization. Int J Radiat Biol 56:35-44
Mullikin, J; Norgren, R; Lucas, J et al. (1988) Fringe-scan flow cytometry. Cytometry 9:111-20
Lucas, J N; Gray, J W (1987) Centromeric index versus DNA content flow karyotypes of human chromosomes measured by means of slit-scan flow cytometry. Cytometry 8:273-9
Gray, J W; Lucas, J; Peters, D et al. (1986) Flow karyotyping and sorting of human chromosomes. Cold Spring Harb Symp Quant Biol 51 Pt 1:141-9
Gray, J W; Langlois, R G (1986) Chromosome classification and purification using flow cytometry and sorting. Annu Rev Biophys Biophys Chem 15:195-235