Construction of genomic libraries in A and cosmid vectors

Abstract A genomic DNA library is a set of cloned fragments of genomic DNA. For most organisms no prior information about the genome is required for library construction (though special methods for DNA isolation may be required). The first genomic libraries of complex genomes were constructed in phage X. vectors cloned in Escherichia coli. This was followed by the introduction of cosmid vectors, plasmids which also use the phage X. packaging system (see Volume I, Chapter 9, Section 3.6). Packaging of recombinant DNA molecules into phage X. heads provides an excellent selection for a minimum insert size. It also specifies a maximum insert size of about 22 kb for a X. vector and 40 kb for a cosmid. More recently, several additional systems for the production of genomic libraries have become available. Pl vectors, bacterial artificial chromosomes (BACs) and Pl arti ficial chromosomes (PACs) (1) are also propagated in E.coli, while yeast artificial chromosomes (YACs) are grown in Saccharomyces cerevisiae (2, 3). All the new vec tors offer significant increases in the size of inserts that can be accommodatedgreater than 100 kb for Pl, up to 300 kb for BACs and PACs, and more than 1 Mb for YACs. These large-insert vectors have the advantage that libraries containing fewer clones are needed in order to cover an entire genome. This greatly facilitates the construction of ordered clone maps and the physical linking of genetic markers. There are, however, some disadvantages inherent to propagating clones of very large DNA fragments in either yeast or E.coli. Not only is there a greater tendency for the DNA to undergo deletions or rearrangements, but the copy number of the cloned DNA also has to be quite low, leading to a relatively low yield of the rather complex insert DNA. Another important practical problem with such libraries is that very high-quality, high-molecular-weight insert DNA, of consistent size, must be prepared prior to cloning. Library construction is therefore laborious and technically demanding.