Machines for automated evolution experiments in vitro based on the serial-transfer concept.

Two machine setups for automated evolution experiments in vitro are described. Both machines enable the monitoring of growing populations of RNA or DNA molecules in real time using high-sensitivity glass fiber laser fluorimeters and an automated sample handling facility for volumes in the microliter range. Growth conditions are kept constant by means of the serial-transfer technique, that is, the successive transfer of a small fraction of a growing population into a fresh solution containing no individuals prior to the transfer. The serial transfer technique was modified to work with large populations and constant growth conditions. In the single-channel evolution machine isothermal amplification reactions (Qbeta-system, 3SR, NASBA, SDA) are monitored successively in single test tubes. This machine is particularly well suited for the investigation of optimal adaptation to altered environmental conditions, as is experimentally demonstrated in the evolution of an RNA quasi-species using ribonuclease A as the selection pressure. The new variant of RNA appeared very rapidly (within approximately 80 generations) without stable intermediates, and it was selected by steadily increasing the RNaseA concentration during the serial-transfer experiment. The other machine, which is described in the second part of this article, is a consequent extension of the single-channel machine, and was designed to allow the multichannel detection of up to 960 samples simultaneously. Thus, high-throughput screening can be applied to evolution experiments. In addition to monitoring isothermal amplification reactions, it is also possible to follow PCR amplifications through thin plastic foils. Initial experiments have demonstrated the suitability of the apparatus for uniformly processing samples and for performing thermocycling.