<P> Most bacteria do not go through a well - defined cell cycle but instead continuously copy their DNA; during rapid growth, this can result in the concurrent occurrence of multiple rounds of replication . In E. coli, the best - characterized bacteria, DNA replication is regulated through several mechanisms, including: the hemimethylation and sequestering of the origin sequence, the ratio of adenosine triphosphate (ATP) to adenosine diphosphate (ADP), and the levels of protein DnaA . All these control the binding of initiator proteins to the origin sequences . </P> <P> Because E. coli methylates GATC DNA sequences, DNA synthesis results in hemimethylated sequences . This hemimethylated DNA is recognized by the protein SeqA, which binds and sequesters the origin sequence; in addition, DnaA (required for initiation of replication) binds less well to hemimethylated DNA . As a result, newly replicated origins are prevented from immediately initiating another round of DNA replication . </P> <P> ATP builds up when the cell is in a rich medium, triggering DNA replication once the cell has reached a specific size . ATP competes with ADP to bind to DnaA, and the DnaA - ATP complex is able to initiate replication . A certain number of DnaA proteins are also required for DNA replication--each time the origin is copied, the number of binding sites for DnaA doubles, requiring the synthesis of more DnaA to enable another initiation of replication . </P> <P> Researchers commonly replicate DNA in vitro using the polymerase chain reaction (PCR). PCR uses a pair of primers to span a target region in template DNA, and then polymerizes partner strands in each direction from these primers using a thermostable DNA polymerase . Repeating this process through multiple cycles amplifies the targeted DNA region . At the start of each cycle, the mixture of template and primers is heated, separating the newly synthesized molecule and template . Then, as the mixture cools, both of these become templates for annealing of new primers, and the polymerase extends from these . As a result, the number of copies of the target region doubles each round, increasing exponentially . </P>

Where does the molecule separate before replication begins