ELSEVIER Journal of Biotechnology 35 (1994) 191-203 journal of biotechnology Application of robotic technology to automated sequence fingerprint analysis by oligonucleotide hybridisation Elmar Maier a,,, Sebastian Meier-Ewert a, Ali R. Ahmadi a, Jon Curtis b, Hans Lehrach a Genome Analysis Laboratory and b Electronics Department, Imperial Cancer Research Fund, P.O. Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, UK Abstract We describe our production line for the rapid analysis of large cDNA libraries applying robotic techniques to automatically pick, amplify, array, hybridise and analyse the clones. We also outline the current state of the hybridisation techniques and describe anticipated future developments of the system. Our approach faces the large-scale analysis of cDNA clones with partial sequence analysis by oligonucleotide fingerprinting in the following way: after picking of individual colonies and arraying them automatically in quadruple density (384-we11) microtitre plates, the cDNA clones are amplified by an automated waterbath polymerase chain reaction (PCR), which allows us to run about 46 000 reactions in parallel. The PCR products are automatically transferred to nylon membranes in a high density pattern using a robotic device. We routinely produce twelve 22 cm x 22 cm membranes in 90 min. Each membrane contains 20 736 clones, although much higher densities might be feasible using both miniaturized glass matrices and fluorescence based hybridisation techniques. Theoretical analysis and preliminary computer simula- tions indicate that about 100-200 sequence specific hybridisations of octanucleotides to about 100 000 PCR products of 1000-1500 base-pairs length will generate sufficient information for classifying the clones into groups of identical or related genes and to identify a large number of previously uncharacterized cDNA clones. Key words: Robotics; Automation; Polymerase chain reaction; Hybridisation; Oligonucleotide fingerprinting; cDNA libaries; Genome analysis 1. Introduction The analysis of large genomes requires the development of rapid, efficient and highly auto- mated procedures for both clone mapping and sequencing. Various elements must come to- gether to accomplish this important task includ- ing the large-scale production and analysis of * Corresponding author. Elsevier Science B.V. SSDI 0168-1656(94)00039-F selected DNA fragments. In our hybridisation- based approach to genome analysis (Lehrach et al., 1990) several steps as, e.g., colony picking, DNA amplification and arraying of clones are repetitious and have so far yielded to automation using robotic techniques which we describe here. A summary of specifications of the robotic sys- tems we have developed in our laboratory is given in Table 1. This automated approach has enabled us to assemble a production line for partial and even-