Rapid arrayed filter production using the 'ORCA' robot.

By adapting a commercially available, general purpose laboratory robot, it is possible to produce high-density gridded hybridization filters of clone colonies or DNA products. We are using this system to produce 60-90, 8 x 12 cm filters in an 8-hour day, each containing 3,456 clones arranged in 96, 6 x 6 grids.

Automated DNA hybridization.

We have developed a system which fully automates the process of membrane-immobilized DNA hybridization. The system consists of three major parts: (i) a membrane-processing cassette which is thermo-controlled and which can hold multiple membranes simultaneously; (ii) a fluidic system which consists of seven independent input channels, a collecting waste container, and flow-controlling valves; and (iii) integrating software which controls the entire system. No pumps are required since the entire fluidic system is gravity driven. The hybridizer is compatible with nonradioactive detection such as chemiluminescence and has been tested on gridded template arrays as part of a large-scale DNA sequencing project.

Arrays of complementary oligonucleotides for analysing the hybridisation behaviour of nucleic acids.

Arrays of oligonucleotides corresponding to a full set of complements of a known sequence can be made in a single series of base couplings in which each base in the complement is added in turn. Coupling is carried out on the surface of a solid support such as a glass plate, using a device which applies reagents in a defined area. The device is displaced by a fixed movement after each coupling reaction so that consecutive couplings overlap only a portion of previous ones. The shape and size of the device and the amount by which it is displaced at each step determines the length of the oligonucleotides. Certain shapes create arrays of oligonucleotides from mononucleotides up to a given length in a single series of couplings. The array is used in a hybridisation reaction to a labelled target sequence, and shows the hybridisation behaviour of every oligonucleotide in the target sequence with its complement in the array. Applications include sequence comparison to test for mutation, analysis of secondary structure, and optimisation of PCR primer and antisense oligonucleotide design.

Microplate capture hybridization of amplified parvovirus B19 DNA fragment labelled with digoxigenin.

A capture hybridization technique in microplate has been developed for the identification of polymerase chain reaction (PCR) amplified B19 DNA fragment in clinical specimens. The amplified 104 bp B19 DNA fragment, located in the gene coding for structural proteins, was directly labelled during the amplification reaction by incorporation of digoxigenin-labelled dUTP. The amplified product was then captured by a probe immobilized on microplate wells. The capture hybridization reaction was visualized as an enzyme-linked immunosorbent assay using anti-digoxigenin Fab fragment labelled with peroxidase. Thirty-five serum samples were tested by our capture hybridization assay and the results were in accordance with the results obtained by Southern blot analysis of PCR amplified product. Our microplate capture hybridization assay showed a high sensitivity and reproducibility and appears to be a practical and reliable test for routine screening of B19 parvovirus DNA in clinical specimens.

Detection of HIV-1 by digoxigenin-labelled PCR and microtitre plate solution hybridisation assay and prevention of PCR carry-over by uracil-N-glycosylase.

An extremely sensitive and convenient microtiter plate solution hybridisation assay for the detection of HIV-1 PCR products was developed. The PCR product is labelled by direct incorporation of digoxigenin-dUTP and after denaturation is captured by a microtitre plate coated with a streptavidin-linked biotinylated probe. The PCR/probe hybrids are reacted with an alkaline phosphate conjugated anti-digoxigenin antibody and detected using an alkaline phosphatase enzyme amplification system. The use of uracil-N-glycosylase and dUTP instead of dTTP in the PCR is used to effectively control carry-over from previous PCR products. The assay can detect single HIV-1 DNA molecules in a background DNA of 0.75 microgram.

[A method of competitive dot hydridization for genotyping influenza A viruses]. / Metod konkurentnoi tochechnoi gibridizatsii dlia genotipirovanii virusov grippa A.

A new method for genetic typing of influenza viruses using molecular hybridization of DNA-RNA was developed which consisted in addition to the hybridization solution, apart from the radioactively labeled probe, of RNA of a virus with known gene homologous to the plasmid DNA used as the probe but belonging to a different serosubtype of influenza A virus, other than cloned kDNA, (within the range of H1N1, H2N2, and H3N2). This competitive RNA (RNAc) is added in considerable excess with regard to both molecular probe and to RNA immobilized on the filter. Therefore hybrids of molecular probe DNA with RNAc are rapidly formed from which RNAc may be replaced only by those bRNA immobilized on the filter whose homology to the probe is higher than that of RNAc.

Automated production of high density cosmid and YAC colony filters using a robotic workstation.

We report here on a system for automated preparation of high-density colony filters of arrayed libraries using the high density replicating system (HDR) for the Beckman Biomek 1000 robotic workstation. This system, consisting of a 96-pin tool, a sterilization station and controlling software, transfers samples from microplates onto target membranes in arrays up to 36 times the density of a 96-well microplate. The transfer operation can be completely automated with the addition of the Biomek Side Loader System, which consists of a robotic arm capable of transferring plates and filters between the Biomek working tablet and a storage area. Using the complete system, we are able to plate 32 replica filters (8 x 12 cm), each containing the clones from 16 different microplates (i.e., 1536 clones per filter), in a 16-h overnight run without any operator intervention. We describe conditions used for transfer of bacterial yeast colonies and fixation of DNA to the membranes, and we illustrate hybridization results obtained with cosmid and YAC filters.