Replication of DNA in mammalian chromosomes: isolation of replicating segments

From Chinese hamster cells, blocked at the beginning of the S phase by treatment with fluorodeoxyuridine and pulse-labeled with [(3)H]thymidine, one can release segments of native DNA containing template polyuncleotide chains and the most recently replicated DNA. The segments, which can be released without shear by lysis at temperatures below the melting point for the double helix, can be separated from both the nascent single chains (Okazaki pieces) and the long strands of native DNA in sodium perchlorate gradients. These 26S segments are equivalent to about 6 mum of native DNA, but may be replication forks rather than linear pieces.