Isolation of human cytomegalovirus DNA from infected cells.

Abstract

Human cytomegalovirus (HCMV) DNA was extracted from infected human embryo fibroblast cultures using the Hirt, Triton-NaCl, and total extraction methods. The Hirt method gave a maximum yield of 60% and was 5- to 10-fold more efficient than the Triton-NaCl method for the extraction of HCMV DNA. However, both methods gave comparable yields ranging from 70 to 75% for the extraction of herpes simplex virus type 1 (HSV1) DNA from infected cells. The Hirt-extracted HCMV DNA contained from 5 to 10% contaminating mitochondrial DNA which could be removed after centrifugation in CsCl-ethidium bromide density gradients. The Hirt-extracted HCMV DNA was analyzed by velocity sedimentation in sucrose gradients and was found to sediment similar to HSV1 55S DNA. Additionally, the Hirt-extracted HCMV DNA was shown to be similar to virion-extracted HCMV DNA in plaquing efficiency and by HindIII restriction digest patterns.