Perng G C, Ghiasi H, Kaiwar R, Nesburn A B, Wechsler S L
Ophthalmology Research Laboratory, Cedars-Sinai Medical Center, Los Angeles, CA 90048.
J Virol Methods. 1994 Feb;46(2):111-6. doi: 10.1016/0166-0934(94)90096-5.
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.
本文描述了使用低拷贝数质粒极大地增强克隆单纯疱疹病毒1型（HSV-1）DNA的能力。HSV-1 DNA的某些区域极难克隆。在病毒的长重复序列和短重复序列中常常会遇到特别的困难。使用普通质粒克隆某些HSV-1序列时，得到的质粒含有异常大小的插入片段和/或比原始质粒小的质粒。这些克隆困难的原因尚不清楚。然而，这些困难可能与HSV-1基因组总体较高的GC含量、重复序列中更高的GC含量、病毒重复序列内的小正向和反向重复序列或未表征的二级DNA结构有关。我们表明，使用低拷贝数质粒pEV-vrf3极大地增强了我们克隆和亚克隆“困难”HSV-1限制片段的能力，包括那些我们无法使用其他质粒克隆的限制片段。
