Detection of extrachromosomal circular DNA sequences from tumor cells by an alkaline lysis, Alu-polymerase chain reaction technique.
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 trends to reflect genetic instability and also suggests that 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 x 10(5) 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 ot aid in their detailed characterizations.
Sen
S
S
Division of Laboratory Medicine, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Rani
S
S
Freireich
E J
EJ
Hewitt
R
R
Stass
S A
SA
D001483:Base Sequence; D014408:Biomarkers, Tumor; D004270:DNA, Circular / Q000032:analysis*; D004273:DNA, Neoplasm / Q000032:analysis*; D005111:Extrachromosomal Inheritance; D006801:Humans; D007938:Leukemia / Q000235:genetics; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods
D014408:Biomarkers, Tumor; D004270:DNA, Circular; D004273:DNA, Neoplasm