In 1987, Bonita J. Brewer and Walton L. Fangman devised a technique known as neutral/neutral two-dimensional gel electrophoresis that locates origins of replication in chromosomes, This technique was used to show that ARSs act as initiation sites for DNA replication in living cells. DNA isolated from unsynchronized rapidly proliferating cells is digested with a restriction endonuclease. Only a small fraction of any given fragment population will be derived from DNA caught in the act of replication and will contain replication forks. These replicating fragments, which vary in size from n to 2n, are first separated according to size by low-voltage agarose gel electrophoresis at neutral pH. Then the lane containing the separated fragments is cut from the gel, rotated 90°, and placed at the top of a new gel for separation in a second dimension.

The new gel contains a higher agarose concentration and is run at high voltage at neutral pH, allowing it to separate fragments by shape, Therefore, fragments that migrated together in the first dimension separate from one another in the second with those containing bubbles migrating at the slowest rate, those with a single replication fork(Y-shaped) at an intermediate rate, and those that are linear duplexes at the fastest rate. Once migration is complete, the fragments are transferred to a positively charged nylon membrane and the migration of a specific fragment is detected by hybridization with a DNA probe that is labeled with a radioactive or fluorescent tag.

Two types of replication intermediates are readily distinguished from the observed patterns. When a replication origin is located within the boundaries of the restriction fragment, the bubble-shaped replication intermediates produce a bubble arc pattern. When a replication fork is passively generated from an origin of replication that is outside the fragment, the Y-shaped replication intermediates generate a Y-arc (probe 2).