A highly sensitive and fast nonradioactive method for detection of polymerase chain reaction products.
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.
Holmstrøm
K
K
Department of Molecular Food Technology, Biotechnological Institute, Lyngby, Denmark.
Rossen
L
L
Rasmussen
O F
OF
D000218:Adamantane / Q000031:analogs & derivatives; D001426:Bacterial Proteins; D001710:Biotin; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin / Q000031:analogs & derivatives; D004800:Enzymes, Immobilized; D006923:Hymecromone / Q000031:analogs & derivatives; D007202:Indicators and Reagents; D008089:Listeria monocytogenes / Q000737:chemistry / Q000235:genetics; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019809:Streptavidin; D013379:Substrate Specificity; D013942:Thymine Nucleotides
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D003856:Deoxyuracil Nucleotides; D004800:Enzymes, Immobilized; D007202:Indicators and Reagents; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D013942:Thymine Nucleotides; C070202:digoxigenin-11-deoxyuridine triphosphate; C061523:3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane; C008644:nitrophenylphosphate; C005359:4-methylumbelliferyl phosphate; D006923:Hymecromone; D001710:Biotin; D019809:Streptavidin; D004076:Digoxigenin; D000218:Adamantane; C024157:thymidine 5'-triphosphate