Establishment of Monitoring System to Detect Single Copy DNA Included in One Genome but not in Another Using Representational Difference Analysis

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Polyrnerase chain reaction (PCR) -coupled subtractive procedure, representa-tional difference analysis (RDA) , is an efficient method to find the differences between two complex genomes. RDA has been applied to detect genetic lesions in cancer, the identification of unknown pathogens from the genomes, and the isolation of polymorphic markers. However, characterization of various clones obtained by RDA is time consuming and laborious work, and it is of great impor-tance to monitor whether RDA really works. To establish a monitoring system to detect single copy target DNA, we studied whether RDA could detect four fragments of non-human DNA which were added in one genome but not in another. We were able to successfully detect the target DNAs which were mixed at the ratio of single and ten copies per haploid genome using RDA with some modification of the original protocol. We confirmed that RDA was sensi-tive and effective enough to detect such genetic lesions as occurred in cancer cells. The target DNA used in this model could be utilized as a positive control in other applications of RDA.

Journal

  • Tumor Research

    Tumor Research 31 21-30, 1996

    Sapporo Medical University

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