Abstract
This chapter describes two methods that are used routinely to assay replication forks. The first method—a modified version of the two-dimensional (2-D) gel method—allows the determination of the direction of fork movement through sequences not containing an origin, as well as determination of origin location and origin efficiency. The second method is an adaptation of the Meselson–Stahl density transfer method that allows the determination of the kinetics of replication for chromosomal restriction fragments of interest. This method—used in concert with the 2-D gel method—provides a way of determining the rate of fork progression through specific segments of the chromosome. The 2-D gel techniques rely on the principle of separating DNA restriction fragments in two dimensions. The DNA of interest must contain branched intermediates; the chapter describes a method to isolate DNA from actively growing yeast cells to enrich for branched replication intermediates.
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