Development and use of metaphase chromosome flow‐sorting methodology to obtain recombinant phage libraries enriched for parts of the human X chromosome

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Abstract
Metaphase chromosomes isolated from human lymphoblastoid cell lines containing structurally abnormal X chromosomes have been stained with the bisbenzimidazole dye Hoechst 33258 and analyzed on a FACS II flow system equipped with a 5‐W all‐lines argon ion laser. The chromosomal fluorescence has been highly resolved at flow rates of 1,000–3,000 chromosomes per second. With the goal of obtaining recombinant DNA libraries from parts of the human X chromosome, fluorescence populations enriched for a dicentric X (Xpter‐> Xq24::Xq24‐> Xpter) chromosome and an isochromosome of the long arm of the X [i(Xq)] have been identified. The dicentric X chromosome has been resolved as a discrete peak in the fluorescence flow histogram. In contrast, the fluorescence intensity of the isochromosome is indistinguishable from that of chromosomes 3 and 4. Recombinant DNA libraries from the flow‐sorted chromosomes have been constructed in the lambda phage, Charon 21A, and consist of 1.6 × 105 and 0.7 × 105 plaque‐forming units in the case of the dicentric X and the isochromosome, respectively. Ninety percent of the phage in both recombinant libraries contain inserts which hybridize to highly repetitive human DNA sequences. The recombinant phage library from the flow‐sorted dicentric X chromosome, which could be assigned to a discrete fluorescence peak, has been further characterized and shows at least a tenfold enrichment for X chromosome‐specific DNA sequences as determined by Southern blot hybridization of cloned fragments.