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.
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