Experimental work on the molecular nature of hereditary material has demonstrated conclusively that DNA (not protein, lipids, or carbohydrates) is indeed the genetic material. Using data obtained by others, Watson and Crick deduced a double-helical model with two DNA strands, wound around each other, running in antiparallel fashion. The binding of the two strands together is based on the fit of adenine (A) to thymine (T) and guanine (G) to cytosine (C). The former pair is held by two hydrogen bonds; the latter, by three.

The Watson–Crick model shows how DNA can be replicated in an orderly fashion—a prime requirement for genetic material. Replication is accomplished semiconservatively in both prokaryotes and eukaryotes. One double helix is replicated to form two identical helices, each with their nucleotides in the identical linear order; each of the two new double helices is composed of one old and one newly polymerized strand of DNA.

The DNA double helix is unwound at a replication fork, and the two single strands thus produced serve as templates for the polymerization of free nucleotides. Nucleotides are polymerized by the enzyme DNA polymerase, which adds new nucleotides only to the 3′ end of a growing DNA chain. Because addition is only at 3′ ends, polymerization on one template is continuous, producing the leading strand, and, on the other, it is discontinuous in short stretches (Okazaki fragments), producing the lagging strand. Synthesis of the leading strand and of every Okazaki fragment is primed by a short RNA primer (synthesized by primase) that provides a 3′ end for deoxyribonucleotide addition.

The multiple events that have to occur accurately and rapidly at the replication fork are carried out by a biological machine called the replisome. This protein complex includes two DNA polymerase units, one to act on the leading strand and one to act on the lagging strand. In this way, the more time-consuming synthesis and joining of the Okazaki fragments into a continuous strand can be temporally coordinated with the less complicated synthesis of the leading strand. Where and when replication takes place is carefully controlled by the ordered assembly of the replisome at certain sites on the chromosome called origins. Eukaryotic genomes may have tens of thousands of origins. The assembly of replisomes at these origins can take place only at a specific time in the cell cycle.

The ends of linear chromosomes (telomeres) present a problem for the replication system because there is always a short stretch on one strand that cannot be primed. The enzyme telomerase adds a number of short, repetitive sequences to maintain length. Telomerase carries a short RNA that acts as the template for the synthesis of the telomeric repeats. These noncoding telomeric repeats associate with proteins to form a telomeric cap. Telomeres shorten with age in somatic cells because telomerase is not made in those cells. Individuals who have defective telomeres experience premature aging.