Strunk G, Ederhof T
Max-Planck-Institute für biophysikalische Chemie, Evolutionsgruppe, Am Fassberg 11, Göttingen, Germany.
Biophys Chem. 1997 Jun 30;66(2-3):193-202. doi: 10.1016/s0301-4622(97)00062-8.
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.
本文描述了两种用于体外自动化进化实验的机器设置。这两种机器都能够使用高灵敏度玻璃纤维激光荧光计和用于微升范围内体积的自动样品处理设备实时监测RNA或DNA分子不断增长的群体。通过连续转移技术保持生长条件恒定，即，在转移之前，将一小部分不断增长的群体连续转移到不含个体的新鲜溶液中。对连续转移技术进行了改进，以适用于大群体和恒定的生长条件。在单通道进化机器中，等温扩增反应（Qβ系统、3SR、NASBA、SDA）在单个试管中连续监测。这台机器特别适合研究对改变的环境条件的最佳适应性，如在以核糖核酸酶A作为选择压力的RNA准种进化中通过实验证明的那样。新的RNA变体出现得非常迅速（在大约80代内），没有稳定的中间体，并且在连续转移实验期间通过稳步增加RNaseA浓度对其进行选择。本文第二部分描述的另一台机器是单通道机器的后续扩展，设计用于同时对多达960个样品进行多通道检测。因此，高通量筛选可应用于进化实验。除了监测等温扩增反应外，还可以通过薄塑料箔跟踪PCR扩增。初步实验证明了该仪器适用于均匀处理样品和进行热循环。
