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Dual-beam high-speed sorting has been developed to facilitate purification of chromosomes based on DNA staining with the fluorescent dyes Hoechst 33258 and chromomycin A3. Approximately 200 chromosomes per second of two types can be sorted from a suspension of chromosomes isolated from human lymphoblasts while fluorescent objects (chromosomes, debris fragments, chromosome clumps, and nuclei) are processed at the rate of about 20,000 per second. This sorting rate is approximately ten times that possible with conventional sorters. Chromosomes of a single type can be sorted with a purity of about 90 percent. DNA from the sorted chromosomes is suitable for construction of recombinant DNA libraries and for gene mapping.

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We report the construction of 15 human chromosome-specific DNA libraries. Metaphase chromosomes were purified by flow sorting and the DNA was extracted and cleaved with Hindlll before cloning into the λ vector Charon 21 A. A sensitive miniblot hybridization method was used to monitor the physical and biochemical steps in the cloning procedure. Using this method, we have developed a highly efficient protocol for producing large numbers of recombinant phage from 0.2–1.0 × 10⁶ sorted chromosomes. DNA from the following chromosomes was cloned: #4, 6, 8, 9, 11, 13, 14+15, 16, 17, 18, 19, 20, 21, 22 and Y. These libraries are available to the scientific research community and will be valuable in the genetic analysis of the human genome.

We have characterized at the molecular level seven chromosome-specific libraries constructed in phage λ Charon 21A from flow-sorted human chromosomes. The purity of libraries prepared from chromosomes sorted from hamster × human cells was estimated by species-specific hybridization and ranged from 48% to 83% of clones containing human inserts. Among libraries of chromosomes from human cells, mass screenings were made for repetitive sequences and 20 clones from the # 18 and # 20 libraries were analyzed in detail. Ten to fifteen percent of all clones contain sequences which can be mapped; 80 100% of these derive from the intended chromosome of origin, demonstrating very high purity and a 35 × enrichment of chromosome-specific sequences over a total genomic library. The two libraries contain a high, though dissimilar, percent of repeat-containing clones; the # 18 library has 55% repetitive clones and the # 20 library 85%. This dissimilarity may be due to a difference in insert size distribution, since the # 18 library has smaller inserts than the # 20. This could be caused by variation in extent of digestion of insert DNA and/or differences in sequence organization between the two chromosomes. A method more sensitive than conventional plaque-lift screening was used to detect repetitive inserts; in this way nearly all repetitive clones could be eliminated before purification of their DNAs.