Metaphase chromosomes with high molecular weight DNA were isolated from Chinese hamster ovary (CHO) cells in a neutral buffer containing polyamines and chelators. The individual, unfixed chromosomes retained their centromeric and secondary constrictions, distinct sister chromatids, and complex banding patterns. The DNA from these chromosomes was 100-fold larger (2 x 10(8) daltons) than DNA from chromosomes isolated by other procedures. These characteristics indicate preservation during isolation of considerable native structure. In contrast to chromosomes produced by other methods, these chromosomes were stable in storage and did not aggregate, thus providing useful material for studies of the structure and biochemistry of individual chromosomes.

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