MACHINES FOR AUTOMATED EVOLUTION EXPERIMENTS IN-VITRO BASED ON THE SERIAL-TRANSFER CONCEPT

Two machine setups for automated evolution experiments in vitro are de scribed. 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 volum es in the microliter range. Growth conditions are kept constant by mea ns of the serial-transfer technique, that is, the successive transfer of a small fraction of a growing population into a fresh solution cont aining no individuals prior to the transfer. The serial transfer techn ique was modified to work with large populations and constant growth c onditions. In the single-channel evolution machine isothermal amplific ation reactions (Q beta-system, 3SR, NASBA, SDA) are monitored success ively in single test tubes. This machine is particularly well suited f or the investigation of optimal adaptation to altered environmental co nditions, 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 generat ions) without stable intermediates, and it was selected by steadily in creasing the RNaseA concentration during the serial-transfer experimen t. The other machine, which is described in the second part of this ar ticle, is a consequent extension of the single-channel machine, and wa s designed to allow the multichannel detection of up to 960 samples si multaneously. Thus, high-throughput screening can be applied to evolut ion experiments. In addition to monitoring isothermal amplification re actions, 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 t hermocycling. (C) 1997 Published by Elsevier Science B.V.