PURE CHROMOSOME-SPECIFIC PCR LIBRARIES FROM SINGLE SORTED CHROMOSOMES

Chromosome-specific DNA libraries can be very useful in molecular and cytogenetic genome mapping studies, We have developed a rapid and simp le method for the generation of chromosome-specific DNA sequences that relies on polymerase chain reaction (PCR) amplification of a single p ow-sorted chromosome or chromosome fragment. Previously reported metho ds for the development of chromosome libraries require larger numbers of chromosomes, with preparation of pure chromosomes sorted by pow cyt ometry, generation of somatic cell hybrids containing targeted chromos omes, or a combination of both procedures. These procedures are labor intensive, especially when hybrid cell lines are not already available , and this has limited the generation of chromosome-specific DNA libra ries from nonhuman species. In contrast, a single sorted chromosome is a pure source of DNA for library production even when flow cytometric resolution of chromosome populations is poor. Furthermore, any sortin g cytometer may be used with this technique. Using this approach, se d emonstrate the generation of PCR libraries suitable for both molecular and fluorescence in situ hybridization studies from individual baboon and canine chromosomes, separate human homologues, and a rearranged m arker chromosome from a transformed cell line. PCR libraries specific to subchromosomal regions have also been produced by sorting a small c hromosome fragment. This simple and rapid technique will allow generat ion of nonhuman linkage maps and probes for fluorescence in situ hybri dization and the characterization of marker chromosomes from solid tum ors. In addition, allele-specific libraries generated by this strategy may also be useful for mapping genetic diseases.