Gc. Perng et al., AN IMPROVED METHOD FOR CLONING PORTIONS OF THE REPEAT REGIONS OF HERPES-SIMPLEX VIRUS TYPE-1, Journal of virological methods, 46(2), 1994, pp. 111-116
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.