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A protocol was designed for the rapid and efficient construction of cosmid libraries from cell-associated viral genomes available in very low quantities. Purification of viral DNA from cellular DNA was unnecessary. The vast excess of cellular DNA compensated for the limited amount of available viral DNA, enabling titration of the restriction endonuclease partial digest. A cosmid library of the turkey herpesvirus DNA genome was constructed from 1.5 micrograms of cellular DNA containing approximately 6 nanograms of viral DNA.

A 1.2-kilobasepair DNA region ( a -like) in the serotype-3 Marek's disease virus was directly repeated at the ends of the long (L) and short (S) components of the genome and repeated in the opposite orientation at the junction between the L and S components. Four variants from plaque-purified virus that vat led in the number of copies of the a-like region were identified. The predominant variant contained one copy at both ends of the genome and at the L-S junction, Two other variants contained either one or two copies at the L end and one to three copies at the L-S junction. The fourth variant lacked the a -like region at the L end and the L-S junction. The fourth variant became the predominate species as the virus was serially passaged in cell culture.

A double-cos-site cosmid vector, c2XMCS, contains a multiple cloning site (MCS) with fifteen restriction sites flanked by phage T7 and T3 RNA polymerase promoters. The two cos sites in the cosmid vector enable the construction of cosmid libraries from unfractionated partial digests of genomic DNA. This simple construct combines the efficiency of a double-cos-site cosmid with the versatility provided by the MCS from pBluescript.