Comparison of self-sustained sequence-replication reaction systems

The 3SR (self-sustained sequence-replication) reaction is a very efficient method for isothermal amplification of target DNA or RNA sequences in vitro. This method requires three enzymatic activities: reverse transcriptase, DNA-dependent RNA polymerase and Escherichia coli ribonuclease H. We have modified the original protocol by using human immunodeficiency virus (HIV)-1 reverse transcriptase instead of avian myeloblastosis virus (AMV) reverse transcriptase to allow amplification with T7 RNA polymerase but without E. coli ribonuclease H. Comparison of the incorporation kinetics between the conventional three-enzyme 3SR and our two-enzyme 3SR shows differences in the kinetic behaviour. Furthermore, by the new two-enzyme 3SR, the amplified RNA is obtained in a purer form compared with the experiments with three-enzyme 3SR. The aim of our research is to adapt 3SR as a useful tool for darwinian evolutionary experiments.