AN IMPROVED METHOD FOR CLONING PORTIONS OF THE REPEAT REGIONS OF HERPES-SIMPLEX VIRUS TYPE-1

The use of a low copy number plasmid to enhance greatly the ability to clone herpes simplex virus type 1 (HSV-1) DNA is described. Certain r egions of the HSV-1 DNA have been extremely difficult to clone. Partic ular difficulties have often been encountered in the long and short vi ral repeats. Attempts to clone certain HSV-1 sequences using common pl asmids produced plasmids containing inserts of unusual size and/or pla smids smaller than the original plasmid. The reason for these cloning difficulties is unclear. However, the difficulties may be related to t he high overall GC content of the HSV-1 genome, the even higher GC con tent of the repeats, small direct and inverted repeats within the vira l repeats, or uncharacterized secondary DNA structure. We show that th e use of the low copy number plasmid pEV-vrf3 greatly enhanced our abi lity to clone and subclone 'difficult' HSV-1 restriction fragments, in cluding restriction fragments that we were unable to clone using other plasmids.