Abstract
Restriction enzyme analysis of CMV DNA is essential for investigating viral transmission. Previous methods for purifying DNA used either prolonged viral passage to obtain cell free virus and/or equilibrium centrifugation in CsCl and/or in vivo 32P labelling and/or DNA transfer to nitrocellulose. Because these methods preclude the rapid inexpensive processing of multiple samples, the following method was used. Following isolation, CMV infected cells were passed to 2-75cm2 petri dishes containing detached MRC-5 cells. When CPE involved >50% of the cells they were lysed using 0.6% SDS. Cellular DNA but not CMV DNA precipitated at 4° using NaCl (Hirt proceedure). After phenol extraction and ethanol precipitation, EcoRI digested CMV DNA was electrophoresised through agarose. DNA bands were detected either with ethidium bromide or by a 2 hour in situ hybridization directly on the agarose gels using as probes 6 32p labelled cloned fragments of the Towne strain. This procedure allows analysis of up to 100 CMV isolates within 3 weeks and purification, digestion, electrophoresis, and/or in Situ hybridization requires only 1 or 2 days. This method will be a valuable adjunet to currently used methods.
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Adler, S., Maurer, H. 1043 A SIMPLE, RAPID, AND SENSITIVE METHOD FOR THE RESTRICTION ENDONUCLEASE ANALYSIS OF THE DNA OF CYTOMEGALOVIRUS (CMV). Pediatr Res 19, 284 (1985). https://doi.org/10.1203/00006450-198504000-01073
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DOI: https://doi.org/10.1203/00006450-198504000-01073