Experimental Proof of DNA Replication

Watson and Crick's semi-conservative model remained an elegant but unproven hypothesis until Matthew Meselson and Franklin Stahl devised an ingenious experiment in 1958 specifically designed to distinguish between all three competing replication models: conservative, dispersive and semi-conservative. They grew two separate cultures of E. coli for many generations, one in a growth medium whose nitrogen source (ammonium chloride) contained the rare, heavy nitrogen isotope 15N, and the other in an otherwise identical medium containing only the common, light isotope 14N, so that essentially every nitrogen atom in each culture's DNA, nitrogen being present throughout the nitrogenous bases, would carry that culture's specific isotope. Because 'heavy' DNA (built from 15N) and 'light' DNA (built from 14N) differ very slightly in mass, they could be physically separated from one another using caesium chloride (CsCl) density-gradient ultracentrifugation, a technique that settles DNA of different densities into distinct, visible bands within a centrifuge tube. Meselson and Stahl then transferred the fully heavy (15N) culture into fresh medium containing only the light isotope (14N), and took DNA samples at fixed 20-minute intervals to track how the density of the bacterial DNA changed as the cells kept dividing and replicating in the new, light medium. After exactly one round of replication, all of the extracted DNA settled into a single band at a density exactly intermediate between the original heavy and light references, showing that each new DNA molecule was a 15N/14N hybrid rather than being either purely heavy or purely light. After a second round of replication, the DNA now separated into two distinct bands of equal intensity: one still at the intermediate, hybrid density, and one now at the fully light density, matching what semi-conservative replication predicts (one 15N strand paired with a newly made 14N strand, or two newly made 14N strands paired together) but ruling o …