Detection of extrachromosomal circular DNA sequences from tumor cells by an alkaline lysis, Alu‐polymerase chain reaction technique

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Abstract
Extrachromosomal circular DNAs ranging in size from submicroscopic molecules of approximately 100 kb to cytogenetically resolvable structures of 1000+ kb called minute and double‐minute chromosomes have been shown to harbor amplified genes in primary tumor cells, tumor cell lines, and drug‐resistant cells grown in vitro. The presence of these molecules in transformed and malignant cells tends to reflect genetic instability and also suggests their role in tumor progression. Using a colon carcinoma cell line, we developed a technique to detect extrachromosomal circular DNA‐specific sequences by Alu‐polymerase chain reaction. Circular DNA was enriched by selective alkaline denaturation of genomic DNA. We have successfully performed this procedure with a minimum of 5 × 105 cells. The technique does not require any prior knowledge of the sequences located on the covalent circular DNA molecules for their detection. The procedure should be useful as a routine screen of primary tumor cells for the presence of extrachromosomal circular DNA and should permit the preparation of specific probes to aid in their detailed characterizations.

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