High throughput screening by multichannel glass fiber fluorimetry

Abstract

As a tool for screening large numbers of biological samples by means of amplification (e.g., Qβ or PCR) we have constructed a thermocycler that includes optionally a 96-channel or 960-channel glass fiber fluorimeter (combined with a cooled charge-coupled-device camera). We briefly describe the system integration of all components like liquid handling, thermostats, an x,y,z robot arm, and the glass fiber fluorimeter. The integrated glass fiber fluorimeter allows sensitive on-line measurements in 960 channels within 5 s. Two different screening procedures were carried out. In a first experiment PCR reactions were done in the presence of the known PCR inhibitor hematin and its suppressor transferrin. The system was used to titrate the suppressor with the inhibitor hematin in order to determine the maximum inhibitor concentration tolerated at a given suppressor concentration. We processed 96 PCR samples in parallel with 11 different concentration steps. In a second experiment the 960-channel glass fiber fluorimeter was used to monitor on line the amplification of the Qβ system in the presence or absence of an inhibitor (heparin). Since the doubling time of Qβ RNA variants is about 20 s, on-line detection is crucial for the experimental setup. The evolution of new RNA species adapted to high inhibitor concentrations could be proved by comparison of the fluorimetric signal and electrophoresis.