A simple and rapid method for preparation of viral DNA from cell associated cytomegalovirus

In the field of human cytomegalovirus pathogenesis there is growing interes t in analyzing recent clinical isolates rather than cell culture adapted la boratory strains. However, true low passage isolates are strictly cell asso ciated prior to cell culture adaptation and only a minor fraction of cells are infected at low passage number. Both conditions hinder the preparation of pure viral DNA. To date, genetic analyses had been carried out mostly wi th supernatant associated cytomegalovirus. A rapid and simple method is des cribed for preparation of viral DNA from low passage cell associated isolat es with little cytopathogenic effect. The protocol is based on a combinatio n of Triton X-100 lysis, nuclease treatment, and subsequent phenol chlorofo rm extraction. Cellular background was reduced significantly to enable clea r detection of all viral DNA fragments in restriction fragment length analy sis. The method yielded DNA which was suitable for downstream applications like cloning of viral DNA fragments or transfection of genomic viral DNA. T his method may facilitate genomic analyses of pathogenic cell associated re cent cytomegalovirus isolates. (C) 1999 Elsevier Science B.V. All rights re served.