APPLICATION OF ROBOTIC TECHNOLOGY TO AUTOMATED SEQUENCE FINGERPRINT ANALYSIS BY OLIGONUCLEOTIDE HYBRIDIZATION

We describe our production line for the rapid analysis of large cDNA l ibraries applying robotic techniques to automatically pick, amplify, a rray, hybridise and analyse the clones. We also outline the current st ate of the hybridisation techniques and describe anticipated future de velopments of the system. Our approach faces the large-scale analysis of cDNA clones with partial sequence analysis by oligonucleotide finge rprinting in the following way: after picking of individual colonies a nd arraying them automatically in quadruple density (384-well) microti tre plates, the cDNA clones are amplified by an automated waterbath po lymerase chain reaction (PCR), which allows us to run about 46 000 rea ctions in parallel. The PCR products are automatically transferred to nylon membranes in a high density pattern using a robotic device. We r outinely produce twelve 22 cm x 22 cm membranes in 90 min. Each membra ne contains 20736 clones, although much higher densities might be feas ible using both miniaturized glass matrices and fluorescence based hyb ridisation techniques. Theoretical analysis and preliminary computer s imulations indicate that about 100-200 sequence specific hybridisation s of octanucleotides to about 100 000 PCR products of 1000-1500 base-p airs length will generate sufficient information for classifying the c lones into groups of identical or related genes and to identify a larg e number of previously uncharacterized cDNA clones.