Herpesvirus genome mapping: a rapid generic approach

A protocol for mapping the genome of the alphaherpesvirus macropodid herpes virus 1 is described. This protocol greatly simplifies a similar protocol t hat was used to map the genome of the poxvirus molluscum contagiosum virus. A single restriction digestion is carried out on the viral DNA, and the fr agments cloned into a plasmid vector. The ends of each cloned fragment are sequenced, translated, and used to search peptide sequence databases. Putat ive genomic maps are constructed by assembling contiguous fragments identif ied by the sharing of common open reading frames and through the demonstrat ed colinearity of herpesvirus genomes belonging to the same subfamily. Olig onucleotide primers designed from the nucleotide sequence at the ends of ea ch cloned fragment enable confirmation of putative contiguous fragments by PCR. Fragments not identified by searches of peptide databases are subclone d using a rapid subcloning method. This approach involves restriction diges tion of the cloned fragment with restriction enzymes present in both the mu ltiple cloning site of the vector, and within the fragment. Digested fragme nts larger than the vector are recircularised and transformed into bacteria to generate subclones for sequence analysis. This subcloning method can al so be used to order rapidly genes within large clones. (C) 1999 Elsevier Sc ience B.V. All rights reserved.