Gregor Sagner

Rapid filter assay for the detection of DNA polymerase activity: direct identification of the gene for the DNA polymerase from Thermus aquaticus.

A method for rapid identification of DNA polymerase activity employing an activated DNA substrate covalently bound to nitrocellulose membranes is described. Samples containing DNA polymerase are spotted and the membranes are incubated in an appropriate polymerization buffer containing radioactively labelled dNTPs. By autoradiography of the dried filters, DNA polymerase activity can be directly identified. The method can be used for fast and large-scale screening of chromosomal expression libraries for heterologous DNA polymerases characterized by activity optima different from those of the host organisms. We have identified the gene of the thermostable DNA polymerase from Thermus aquaticus in an expression library of Escherichia coli.

Use of platinum coproporphyrin and delayed luminescence imaging to extend the number of targets FISH karyotyping

Combinatorial use of fluorophores in multicolor fluorescence in situ hybridization (FISH) allows for the recognition of all human chromosomes. Here we introduce the concept of the use of delayed luminescence labels such as phosphorescent platinum coproporphyrins (PtCP) to extend the number of simultaneously detectable targets in multicolor FISH karyotyping. PtCP-conjugated antibodies were used in combination with conventional FISH labels such as cascade blue, fluorescein, lissamine rhodamine, Cy5, and Cy7. Probe sets for all human chromosomes were generated and labeled with these dyes in a combinatorial approach. Delayed luminescence of PtCP was accomplished using a standard fluorescence microscope in which a specially constructed module for visualization of delayed luminescence was incorporated. The module consists of a minichopper incorporated in the standard block that holds the shutter and diaphragm, and a FLC polarizing shutter mounted in a filter holder at the emission side. Multicolor FISH staining was applied to normal metaphase chromosomes and to chromosomes generated from cultured JVM-2 cells with known translocations. Multicolor FISH images (conventional and delayed) were registered using a slow-scan CCD camera. Recognition of all 24 chromosomes was feasible, since the delayed PtCP fluorescence (lifetime, 90 micros) could be easily distinguished from the conventional promptly fluorescing dyes. We discuss possibilities for extending the number of targets far beyond the 24 demonstrated so far.

The Digoxigenin: Anti-Digoxigenin (DIG) System

The digoxigenin:anti-dioxigenin (DIG) indicator system is based on the specific interaction between the cardenolide steroid DIG, a chemically derived aglycon of digoxin and lanatoside C (see figure), and a high-affinity, DIG-specific antibody (Kessler, 1991).

Labeling of Specific DNA Sequences with Digoxigenin during Polymerase Chain Reaction

A broad spectrum of methods for the detection of specific nucleic acids is applied in molecular biology, genetics and medicine.

Blot Formats: Nucleic Acids

Binding between analyte and modified probe, resulting in stable hybrid molecules, is most prominently mediated by specific interaction between complementary purine and pyrimidine bases forming A:T and G:C base pairs.