Isolation of Epstein-Barr Virus in Gastric Cancer of a Young Adult by Efficiency-Monitored Representational Difference Analysis

Gastric cancer in young adults is one of the most mortal neoplasms and is characterized by histopathological features of poorly differentiated type and infiltrative growth. However, very little is known about the genetic mechanisms of this malignant tumor, which is difficult to analyze due to the proliferation of scirrhous stroma. The aim of this study was to apply an improved representational difference analysis （RDA） in order to identify the genetic alterations in gastric cancer of young adults. To monitor the efficiency of RDA, mouse DNA fragment was added to a tester prepared from the genomic DNA of gastric cancer of a 24-yr-old woman, at the rate of a single copy per haploid genome （efficiency-monitored RDA （EM-RDA））. The difference products were analyzed by Southern blot with the mouse DNA as a probe. The real difference products, other than the mouse DNA, were subcloned and sequenced. As a result, added control DNA fragments were sufficiently amplified through 3 rounds of subtraction and the high efficiency of the subtraction was confirmed. Another two difference clones were identified as DNA fragments of Epstein-Barr virus （EBV）. The existence of EBV in the gastric cancer cells was proved by EBV-encoded small RNA 1 in situ hybridization. In summary, efficiency-monitored RDA was demonstrated to raise the reliability of RDA. Furthermore, this method successfully contributed to the detection of EBV in the gastric cancer of a young woman.departmental bulletin pape