Construction and characterization of chromosomal DNA libraries.

Abstract

The construction of an extensive human genetic linkage map will require the generation of large numbers of DNA probes specific for single chromosomes. Recombinant DNA libraries representing chromosomes 22 and 21 have been constructed with a view to studying the specific rearrangements of chromosome 22 observed in chronic myeloid leukaemia and Burkitt’s lymphoma and also the association of chromosome 21 with Down’s syndrome. This was accomplished by sorting about 2 × 106 copies of chromosome 22 and 21 by flow cytometry and insertion of the DNA into the vector λgt WESλB. Twenty clones selected at random from the chromosome 22 library hybridised to EcoRI-digested human DNA, and five of these hybridised to single bands identical in size to the phage inserts. Altogether six single-copy sequences and a clone coding for an 8S RNA isolated by screening the chromosome 22 library for expressed sequences were characterised in detail. Hybridisation of all seven clones to a panel of sorted chromosomes and hybrid cell lines confirmed the assignment of the sequences to chromosome 22. The sequences were localised to regions of chromosome 22 by hybridisation to translocated chromosomes sorted from a cell line having a balanced translocation t(17;22)(p13;q11) and to hybrid cell lines containing the various portions of another translocation t(X;22)(q13;q112). Five clones reside on the long arm of chromosome 22 between q112 and qter, while two clones and an 18S rRNA gene isolated from the chromosome 22 library reside between pter and q112. In situ hybridisation has further mapped one of these clones to the region q13-qter. An identical approach was used for chromosome 21 and three out of five single copy clones have been identified as specific to this chromosome by hybridisation to DNA from a cell hybrid containing only human chromosome 21. In summary, it has been shown that this approach has yielded DNA libraries of high purity based on chromosomes 21 and 22. This method has the advantage of being direct and applicable to nearly all human chromosomes and will be important in the molecular analyses of human genetic disease.