hymecromone and 3-(2--spiroadamantane)-4-methoxy-4-(3---phosphoryloxy)phenyl-1-2-dioxetane

A novel system for the detection of polymerase chain reaction (PCR) products has been developed. The system is based on a PCR in which one of the primers is biotinylated and digoxigenin-11-dUTP is incorporated during elongation. Biotinylated PCR products are captured on streptavidin-coated solid supports, and alkaline phosphatase conjugated to anti-digoxigenin antibody is subsequently bound to the incorporated digoxigenin. The detection may be obtained with colorimetric, fluorescent, or luminescent substrates for alkaline phosphatase. The detection system can be performed in microtiter plates allowing easy handling of multiple samples. The total assay time following the PCR is between 1 and 2 h dependent on the type of substrate and the type of solid support applied in the system. Within this period of time the system is capable of detecting 1 template in 29 cycles of PCR.

Topics: Adamantane; Bacterial Proteins; Biotin; Deoxyuracil Nucleotides; Digoxigenin; DNA, Bacterial; Enzymes, Immobilized; Hymecromone; Indicators and Reagents; Listeria monocytogenes; Nitrophenols; Organophosphorus Compounds; Polymerase Chain Reaction; Sensitivity and Specificity; Streptavidin; Substrate Specificity; Thymine Nucleotides

A method for measuring the amount of a nonradiolabeled DNA probe using four detection substrates is described. In preliminary experiments, digoxygenin-labeled DNA was bound to neutral, nylon membranes and detected with anti-digoxygenin antibodies conjugated to alkaline phosphatase. Four substrates [4-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, AttoPhos, and adamantyl 1, 2-dioxetane phosphate (AMPPD)] were assessed for use in a quantitative hybridization assay. Only AttoPhos and AMPPD were found to have detection limits in the low picogram range and to respond linearly to DNA concentrations ranging from 0 to 1250 pg. In subsequent experiments, a 200-bp DNA probe cloned from the marine bacterium Pseudomonas perfectomarina 23S rRNA gene was hybridized to P. perfectomarina genomic DNA and total RNA. The amount of hybridized probe was determined using AttoPhos. Finally, a digoxygenin-labeled oligonucleotide was probed against genomic DNA. Linearity with respect to DNA concentration was observed using both the 200-bp fragment and the oligonucleotide as probes with a final target detection limit of 166 fg. This study demonstrates the substrate AttoPhos can be used to quantify the amount of nonradiolabeled probe hybridized to target with sufficient sensitivity for very dilute samples, such as environmental samples.

Topics: Adamantane; Alkaline Phosphatase; Base Sequence; DNA Probes; Fluorescent Dyes; Hymecromone; Membranes, Artificial; Molecular Sequence Data; Nitrophenols; Nucleic Acid Hybridization; Organophosphorus Compounds