Rapid Dot Blot Quantitation of Viral DNA and Amplified Genes in Less than 1,000 Human Cells

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Abstract
The copy number of intracellular DNA sequences can be quantitated rapidly with great sensitivity in 100 to 1,000 cells as starting material. The method applies DNA from lysed cells to a charged nylon membrane that permits successive hybridizations with probes for different genes or DNA sequences. This method was tested with eight types of human cells, including leukemic cells, and has detected Epstein-Barr virus (DNA virus) in immortalized cells, integrated HTLV-I (RNA retrovirus) in infected cells, and determined copy numbers of the amplified multiple drug-resistant gene in human cells resistant to various cytotoxic agents. It could also be used for estimating copy number of transfected DNA in human or other mammalian cells. The described method is not as sensitive as polymerase chain reaction may potentially prove, but is easily quantitated for accurate clinical diagnosis where sensitive and quantitative assays must be carried out on a limited number of cells. Examples of the method's clinical application are the staging of human neuroblastomas and the evaluation of oncogene amplification, which has prognostic value for both overall survival and relapse time in breast cancer patients.