Another method of copying DNA, sometimes used in vivo by bacteria and viruses, is the process of rolling circle replication.[20] In this form of replication, a single replication fork progresses around a circular molecule to form multiple linear copies of the DNA sequence. In cells, this process can be used to rapidly synthesize multiple copies of plasmids or viral genomes.
In the cell, rolling circle replication is initiated by an initiator protein encoded by the plasmid or virus DNA. This protein is able to nick one strand of the double-stranded, circular DNA molecule at a site called the double-strand origin (DSO) and remains bound to the 5' phosphate end of the nicked strand. The free 3' hydroxyl end is released and can serve as a primer for DNA synthesis. Using the unnicked strand as a template, replication proceeds around the circular DNA molecule, displacing the nicked strand as single-stranded DNA. Continued DNA synthesis produces multiple single-stranded linear copies of the original DNA in a continuous head-to-tail series. In vivo these linear copies are subsequently converted to double-stranded circular molecules.
Rolling circle replication can also be performed in vitro and has found wide uses in academic research and biotechnology, often used for amplification of DNA from very small amounts of starting material. Replication can be initiated by nicking a double-stranded circular DNA molecule or by hybridizing a primer to a single-stranded circle of DNA. The use of a reverse primer (or random primers) produces hyperbranched rolling circle amplification, resulting in exponential rather than linear growth of the DNA molecule.