Herpesvirus genome mapping: a rapid generic approach
1999
Thomson, Darelle | Smith, Greg
A protocol for mapping the genome of the alphaherpesvirus macropodid herpesvirus 1 is described. This protocol greatly simplifies a similar protocol that 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 fragments cloned into a plasmid vector. The ends of each cloned fragment are sequenced, translated, and used to search peptide sequence databases. Putative genomic maps are constructed by assembling contiguous fragments identified by the sharing of common open reading frames and through the demonstrated colinearity of herpesvirus genomes belonging to the same subfamily. Oligonucleotide primers designed from the nucleotide sequence at the ends of each cloned fragment enable confirmation of putative contiguous fragments by PCR. Fragments not identified by searches of peptide databases are subcloned using a rapid subcloning method. This approach involves restriction digestion of the cloned fragment with restriction enzymes present in both the multiple cloning site of the vector, and within the fragment. Digested fragments larger than the vector are recircularised and transformed into bacteria to generate subclones for sequence analysis. This subcloning method can also be used to order rapidly genes within large clones.
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