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Highly Efficient Method for Obtaining a Subtracted Genomic DNA Library by the Modified In-Gel Competitive Reassociation Method¹

Genetics Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104 [H. S., N. A., A. T., T. S., M. T.]; Department of Pathology, Osaka University Medical School, 1-3, Yamadagaoka, Suita City, Osaka 565 [S. N.]; Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1, Yayoi 1-chome, Bunkyou-ku, Tokyo 113 [M. O.], Japan

A highly efficient method to obtain a subtracted genomic DNA library using 1 µg of target DNA was developed by modification of the previously reported in-gel competitive reassociation procedure. The modified method was based on polymerase chain reaction amplification after selective purification of a target-target reassociated molecule of subtracted DNA fragments to increase cloning efficiency.

For a model experimental system, the subtracted DNA library was constructed after two cycles of subtractive reassociation between cervical cancer DNA fragments containing human papilloma virus DNA and the 100-fold excess of dephosphorylated normal tissue DNA fragments which were size-fractionated in agarose gel. Colony hybridization using human papilloma virus DNA as a probe revealed that a more than 500-fold enrichment of human papilloma virus DNA sequences in the subtracted DNA library could easily be obtained. This simple and efficient method will enable us to isolate an unknown foreign DNA fragment and an unknown amplified DNA fragment which might be present in cancer.

¹ Supported in part by a Grant-in-Aid for A Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan; by Grants-in-Aid from the Ministry of Health and Welfare and from the Ministry of Education, Science and Culture of Japan; by the Bristol-Myers Squibb Foundation; and by the Uehara Memorial Foundation.

² To whom requests for reprints should be addressed.

Received 7/21/94. Accepted 10/ 6/94.

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