Selective capture of acentric fragments by micronuclei provides a rapid method for purifying extrachromosomally amplified DNA.
The amplification and overexpression of a number of oncogenes is strongly associated with the progression of a variety of different cancers. We now present a strategy to purify amplified DNA on double minute chromosomes (DMs) to enable analysis of their prevalence and contribution to tumourigenesis. Using cell lines derived from four different tumour types, we have developed a general and rapid method to purify micronuclei that are known to entrap extrachromosomal elements. The isolated DNA is highly enriched in DM sequences and can be used to prepare probes to localize the progenitor single copy chromosomal regions. The capture of DMs by micronuclei appears to be dependent on their lack of a centromere rather than their small size.
Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA.
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Shimizu
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Kanda
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GM
Wahl
D002458:Cell Fractionation / Q000379:methods; D002503:Centromere / Q000648:ultrastructure; D002869:Chromosome Aberrations; D015342:DNA Probes; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005784:Gene Amplification; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D017404:In Situ Hybridization, Fluorescence; D048629:Micronuclei, Chromosome-Defective / Q000648:ultrastructure*; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D004791:Enzyme Inhibitors; D012264:Ribonucleotide Reductases; D006918:Hydroxyurea