Chromosome-specific gridded cosmid libraries: construction, handling, and use in parallel and integrated mapping

Abstract The analysis of the information in human genomic DNA is based on the cloning and molecular characterization of the genes together with the genetic analysis of the phenotypes a-;sociated with the genetic loci under study. Libraries of cloned genomic DNA serve as essential intermediates to relate the information from the genetic analysis of phenotypic variation to the molecular analysis of the genes (I). Such libraries, can be either constructed in a yeast or bacterial vector-host system. The yeast system. using yeast artificial chromosomes (YACs) (2) as vectors, is well suited for rapid coverage of long genomic regions and entire chromosomes (3) (see also Chapter 4). This offers easier integration of the data with the genetic and physical maps of the genomic DNA, for example in the comparison of the maps of cloned DNA to long-range restriction maps of genomic DNA constructed using restriction endonucleases that cut at infrequent intervals. The yeast system is however not the best choice for the application of methods to identify transcribed regions (genes), to analyse the transcription unit and exon-intron organization, and to obtain the sequence of the genes. The longer the YAC clones arc, the more frequently they suffer from deletions, rearrangements, co-cloning, and chimerism, which makes the establishment of a highresolution map difficult. Moreover, irrespective of the YAC length, the isolation of pure YAC DNA from the genomic DNA of the host is difficult to achieve in most cases (sec Chapter 4 for a discussion of these problems). The bacterial (E. coli)-based vector systems (phage, cosmid, Pl, F-) (4-6) are much more suitable for the characterization of transcribed sequences (7-10), to study the molecular organization of the genes (11), and to provide material for DNA sequencing. The isolation of the cloned DNA in a pure state, free from the DNA of the host, is much more straightforward. Cosmid libraries are probably the simplest to use. They can be constructed with very high efficiency, allowing libraries to be made from limited amounts of DNA, and have been in use for longer than the Pl, PAC, and other new bacterial vector systems.