DNA replication begins at specific sites on a chromosome called origins of replication. These are specific DNA sequences that serve as a starting point for the synthesis of new DNA strands. Origins of replication are typically found in prokaryotes (bacteria and archaea) and are usually several hundred base pairs long. In eukaryotes (animals, plants, fungi, and protists), origins of replication are typically shorter and more numerous, and replication may begin at multiple sites along a chromosome simultaneously.

During replication, the DNA molecule unwinds at the replication fork, and the two strands serve as templates for the synthesis of new complementary strands. The enzyme helicase unwinds the double helix and separates the two strands of DNA, and the enzyme primase synthesizes short RNA primers on the template strands. These primers provide a starting point for the synthesis of the new DNA strands by the enzyme polymerase. As polymerase synthesizes the new DNA strands, it extends the RNA primers with deoxyribonucleotides (the building blocks of DNA). Once the new DNA strands are synthesized, the RNA primers are removed and replaced with DNA by the enzyme ligase.