Effects of Treatment on Differential Staining of BrdU Labeled Metaphase Chromosomes: Three-way Differentiation of M3 Chromosomes

In this paper methodology is described which yields three-way Giemsa differentiation (light-medium-dark) in human metaphase chromosomes exposed to 5-bromodeoxyuridine (BrdU) for 3 DNA synthetic periods (or exposed for 2 DNA synthetic periods and removed from exposure for the third) by means of which all of the sister chromatid exchanges (SCEs) occurring during (or shortly after) S1, S2 and S3 can be accurately counted and distinguished from one another. Using these methods it has been demonstrated that approximately twice as many SCEs occur during the first S-period in the presence of the drug (labeling=B1T0XT0B1)1 as occur during the second S-period (labeling=B2B1XT0B2)1. The three-way differentiation pattern is thought to result from a stepwise decrease in the amount of BrdU incorporated during the first, second and third DNA synthetic periods. These methods can also be used to differentiate between unlabeled (T2T0) and unifilarly labeled (B1T2) sister chromatids and are potentially useful in the detection of sub-chromatid exchanges (none were detected).