부산대학교 도서관

We describe conditions for rolling-circle amplification (RCA) of individual DNA molecules 5-7 kb in size by >[10.sup.9]-fold, using [phi] 29 DNA polymerase. The principal difficulty with amplification of small amounts of template by RCA using [phi] 29 DNA polymerase is 'background' DNA synthesis that usually occurs when template is omitted, or at low template concentrations. Reducing the reaction volume while keeping the amount of template fixed increases the template concentration, resulting in a suppression of background synthesis. Cell-free cloning of single circular molecules by using [phi] 29 DNA polymerase was achieved by carrying out the amplification reactions in very small volumes, typically 600 nl. This procedure allows cell-free cloning of individual synthetic DNA molecules that cannot be cloned in Escherichia coil, for example synthetic phage genomes carrying lethal mutations. It also allows cell-free cloning of genomic DNA isolated from bacteria. This DNA can be sequenced directly from the [phi] 29 DNA polymerase reaction without further amplification. In contrast to PCR amplification, RCA using [phi] 29 DNA polymerase does not produce mutant jackpots, and the high processivity of the enzyme eliminates stuttering at homopolymet tracts. Cell-free cloning has many potential applications to both natural and synthetic DNA. These include environmental DNA samples that have proven difficult to clone and synthetic genes encoding toxic products. The method may also speed genome sequencing by eliminating the need for biological cloning. DNA sequencing | rolling-circle amplification | synthetic DNA