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 regions of the HSV-1 DNA have been extremely difficult to clone. Particular difficulties have often been encountered in the long and short viral repeats. Attempts to clone certain HSV-1 sequences using common plasmids produced plasmids containing inserts of unusual size and/or plasmids smaller than the original plasmid. The reason for these cloning difficulties is unclear. However, the difficulties may be related to the high overall GC content of the HSV-1 genome, the even higher GC content of the repeats, small direct and inverted repeats within the viral repeats, or uncharacterized secondary DNA structure. We show that the use of the low copy number plasmid pEV-vrf3 greatly enhanced our ability to clone and subclone 'difficult' HSV-1 restriction fragments, including restriction fragments that we were unable to clone using other plasmids.