Construction and characterization of three region-specific microdissection libraries for human chromosome 18

Abstract

Three region-specific libraries for the entire human chromosome 18 were constructed using microdissection and MboI linker-adaptor microcloning techniques. The libraries included 18pter-p11.1 (designated 18P library), 18q11.1-q12.3 (18Q1 library), and 18q21.1-qter (18Q2 library). Samples of the microclones from each library were analyzed in detail. The insert sizes ranged between 50-600 bp, with a mean of 180-220 bp for the three libraries. The libraries contained approximately 40-60% microclones with unique sequence inserts. More than 30 unique sequence microclones from each library were analyzed by Southern blot hybridization to demonstrate that they are human specific and were derived from chromosome 18. The human genomic HindIII fragments hybridized to each microclone were determined and microclones cross-hybridized to rodent species were identified. These region-specific libraries and the unique sequence microclones from the libraries are useful reagents for (1) isolating highly polymorphic microsatellite markers for refined linkage analysis, (2) identifying corresponding YAC, BAC or other clones with large inserts for contig assembly and high resolution physical mapping, (3) isolating cDNA clones from the dissected region, and (4) convenient sequencing of the microclones to prepare high density markers and sequence-tagged sites (STSs). Such applications have been demonstrated in a series of similarly constructed microdissection libraries from other regions of the human genome.