Application of robotic technology to automated sequence fingerprint analysis by oligonucleotide hybridisation.
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-well) 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 simulations 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.
Maier
E
E
Genome Analysis Laboratory, Imperial Cancer Research Fund, London, UK.
Meier-Ewert
S
S
Ahmadi
A R
AR
Curtis
J
J
Lehrach
H
H
D001709:Biotechnology; D016172:DNA Fingerprinting / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D015894:Genome, Human; D006801:Humans; D007091:Image Processing, Computer-Assisted; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D012371:Robotics / Q000295:instrumentation* / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides