STRAND DISPLACEMENT AMPLIFICATION AS AN IN-VITRO MODEL FOR ROLLING-CIRCLE REPLICATION - DELETION FORMATION AND EVOLUTION DURING SERIAL TRANSFER

Strand displacement amplification is an isothermal DNA amplification r eaction based on a restriction endonuclease nicking its recognition si te and a polymerase extending the nick at its 3' end, displacing the d ownstream strand. The reaction resembles rolling-circle replication of single-stranded phages and small plasmids. The displaced sense strand serves as target for an antisense reaction and vice versa, resulting in exponential growth and the autocatalytic nature of this in vitro re action as long as the template is the limiting agent. We describe the optimization of strand displacement amplification for in vitro evoluti on experiments under serial transfer conditions. The reaction was foll owed and controlled by use of the fluorescent dye thiazole orange bind ing to the amplified DNA. We were able to maintain exponential growth conditions with a doubling time of 3.0 min throughout 100 transfers or approximate to 350 molecular generations by using an automatic handli ng device. Homology of in vitro amplification with rolling circle repl ication was mirrored by the occurring evolutionary processes. Deletion events most likely caused by a slipped mispairing mechanism as postul ated for in vivo replication took place. Under our conditions, the mut ation rate was high and a molecular quasi-species formed with a mutant lacking internal hairpin formation ability and thus outgrowing all ot her species under dGTP/dCTP deficiency.