Nucleic acid spot hybridization: rapid quantitative screening of lymphoid cell lines for Epstein-Barr viral DNA

Abstract

A simple nucleic acid hybridization method to screen numerous samples of eukaryotic cells rapidly for their Epstein-Barr virus (EBV) DNA content is described. Whole cells are spotted on nitrocellulose filters and their DNA is denatured and fixed to the filter. The resultant DNA spots are hybridized to nick-translated EBV DNA and the extent of hybridization is monitored by autoradiography and scintillation counting. Statistical analysis of serial dilutions of cells permits their viral genome content to be estimated quantitatively by reference to a known standard, such as Raji cells or an artificial mixture of pure viral DNA and uninfected lymphocytes. The sensitivity of the method is between 5 and 50 pg of viral DNA. With this method we are able to select subclones that are high produces of EBV DNA and to identify the optimal time for harvest of EBV DNA from cultured cells. Spot hybridization should permit any cell population or fluid to be screened for the presence of a DNA sequence for which a radioisotopically labeled probe is available.