Enhanced chemiluminescence in filter-based DNA detection.
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DNA↗
Biomedical subjects
I Durrant
Publications and source records attributed to I Durrant.
Biotinyl and phosphotyrosinyl phosphoramidite derivatives useful in the incorporation of multiple reporter groups on synthetic oligonucleotides.
Non-nucleosidic phosphoramidite linker units suitable for use on commercial DNA synthesis machines have been designed for the direct incorporation of biotin and a new reporter group, phosphotyrosine, at multiple sites on synthetic oligonucleotides. The units are based on a 3-carbon glyceryl backbone where the reporter group is attached to the 2-O-position through a 3-aminopropyl spacer. 17-mer oligonucleotides were synthesized carrying at the 5'-end 1, 2, 4 or 8 biotinyl units or 1, 2, 4 or 8 phosphotyrosinyl units respectively and used for the detection of DNA on nitrocellulose filters by hybridization. Subsequent incubation of the filters with a monoclonal antibody to the reporter group followed by secondary detection using enhanced chemiluminescence (ECL) resulted in amplification of signal strengths as the number of reporter groups was increased. The results were quantitated by use of a charge couple device (CCD) camera. Spacing of biotin moieties by thymidyl residues resulted in further improvements in signal strengths, whereas similar spacing of phosphotyrosinyl units did not.
Base Sequence↗
Light-based detection of biomolecules.
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Blotting, Western↗
The application of enhanced chemiluminescence to membrane-based nucleic acid detection.
The ECL gene detection system is a novel, sensitive, non-radioactive system for the detection of nucleic acid hybridized on both nylon and nitrocellulose membranes. It is characterized by direct labeling of probe sequences with horseradish peroxidase combined with an enhanced chemiluminescent (ECL) detection reaction; the light output is captured on blue-light sensitive film. The application of the system to a range of standard molecular biology hybridization techniques is described.
Blotting, Northern↗
Effect of glycerol on thermolysin-catalyzed peptide bond synthesis.
The effect of glycerol on the hydrolytic activity of thermolysin (EC 3.4.24.4) has been compared with the effect on the condensation of N-benzyloxycarbonyl-L-aspartic acid with L-phenylalanine methyl ester to form N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester (Z X Asp X Phe X OMe), the precursor to the sweet-tasting compound L-aspartyl-L-phenylalanine methyl ester. Hydrolytic activity was measured by the degradation of azocasein and furylacryloyl-L-glycyl-L-leucinamide. Increasing concentrations of glycerol reversibly inhibited the hydrolytic activity of the enzyme toward both substrates. The inclusion of glycerol in the synthetic medium facilitated the production of Z X Asp X Phe X OMe in a water-soluble system but reduced the initial rate of peptide synthesis. Glycerol stabilized thermolysin against thermal denaturation.
Catalysis↗
Comparison of seven bio- and chemiluminescent reagents for in situ detection of antigens and nucleic acids.
Bio- and chemiluminescence have proved sensitive enough to compete with chromogenic and radioisotopic tracers for in situ detection. However, they must also provide a discriminant morphological analysis of the specific signal. We have tested seven bio- or chemiluminescent reagents for tissue antigen and nucleic acid detection by immunocytochemistry (ICC) or in situ hybridization (ISH). They were based on luminescent detection of peroxidase, alkaline phosphatase, beta-galactosidase or xanthine oxidase. We also explored whether high molecular weight polymers could increase the spatial definition of the photon emission. An ICCD camera was used to collect the light signal provided by immunolabelling of endothelial cells and by ISH of human papilloma virus on cell smears. Among the enzyme-luminescent substrate combinations tested, the enhanced luminol chemiluminescence (ECL) gave the best resolution of the specific signal. The other systems were mainly hampered by a high diffusion of the reaction product over the tissue section. Unfortunately, in this case, the high molecular weight polymers tested were inefficient. However, the addition of polyvinylalcohol (PVA) or polyvinylpyrrolidone (PVP) significantly improved respectively the definition and intensity of ECL photon emission. We demonstrate that chemiluminescence gives a morphological resolution allowing histological examination. The extension of this new application, now depends on physicochemical adaptation of chemiluminescent reagents to the constraints of tissue detection.
Antigens↗