ABSTRACT
Oligonucleotide fingerprinting is an attractive, high-throughput complement to tag sequencing methods to determine the spectrum and abundance of genes in cDNA libraries. This method currently relies on the sequential hybridizations of short, radioactively labeled DNA oligonucleotides to clone arrays. Here, we describe a new environment that substantially improves this technology. Fluorescently labeled peptide nucleic acid (PNA) oligonucleotides are used as hybridization probes. Hybridization results are recorded with a large-field, high-resolution laser scanner developed for this purpose. Automated image analysis allows easy handling of large numbers of hybridization images. Signal interference effects, which limit the gridding density in the radioactive approach, are strongly reduced. The sensitivity of the fluorescence detection demonstrated permits the convenient use of nylon membranes. Hybridization data quality is improved, and its generation is substantially accelerated, simplified, and less expensive.
Subject(s)
DNA Fingerprinting , Oligonucleotides/chemistry , Autoanalysis , DNA Probes , DNA, Complementary , Fluorescent Dyes , Nucleic Acid Hybridization , Peptide Nucleic Acids , Phycoerythrin , Polymerase Chain Reaction , Reproducibility of ResultsABSTRACT
The usage of standard 96 well microplates for the screening of crystallization conditions of recombinant proteins offers several advantages when compared to commonly used crystallization plate formats. The adoption of robotic technology for plate and glass slide preparation within a "hanging drop" vapour diffusion crystallization experiment enables to work with an increased throughput at reduced costs. In addition to commercial pipetting devices with a 96-channel aspirator/dispenser, solenoid ink-jet technology was applied to form 250 nl droplets with a diameter of approximately 1 mm. This allows miniaturization of crystallization screening set-ups with an estimated ten-fold cost reduction when compared to commonly used 24 well plates.
Subject(s)
Crystallography/instrumentation , Crystallography/methods , Miniaturization/instrumentation , Miniaturization/methods , Recombinant Proteins/chemistry , Concanavalin A/chemistry , Crystallization , Indicators and Reagents , Muramidase/chemistry , SolutionsABSTRACT
We have used oligonucleotide-fingerprinting data on 60,000 cDNA clones from two different mouse embryonic stages to establish a normalized cDNA clone set. The normalized set of 5,376 clones represents different clusters and therefore, in almost all cases, different genes. The inserts of the cDNA clones were amplified by PCR and spotted on glass slides. The resulting arrays were hybridized with mRNA probes prepared from six different adult mouse tissues. Expression profiles were analyzed by hierarchical clustering techniques. We have chosen radioactive detection because it combines robustness with sensitivity and allows the comparison of multiple normalized experiments. Sensitive detection combined with highly effective clustering algorithms allowed the identification of tissue-specific expression profiles and the detection of genes specifically expressed in the tissues investigated. The obtained results are publicly available (http://www.rzpd.de) and can be used by other researchers as a digital expression reference.