Ultrasensitive chemiluminescent and colorigenic detection of DNA, RNA, and proteins in plant molecular biology.
Nonradioactive detection methods for DNA, RNA, and protein analysis have been the subject of research for several years. In this paper the application of the digoxigenin nucleic acid labeling system, in combination with the new alkaline phosphatase substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)-phenyl -1,2-dioxetane, to the special requirements of the analysis of transgenic plants is described. Earlier detection systems lacked the required ultrasensitive limits of detection necessary because of the large genomes found in plant cells. Routine detection of single-copy genes from transgenic plant species requires the detection of bands of picograms of specific DNA, which is easily achieved by employing the AMPPD substrate. Optimal conditions of genomic Southern analysis have been successfully adapted for Northern blotting techniques. Detection of foreign proteins in transgenic plants has proven difficult because of the very small amounts of detectable specific protein. Until now, utilization of biotinylated antibodies in combination with a streptavidin-alkaline phosphatase conjugate has been the most sensitive procedure. By introducing the AMPPD substrate, a further significant enhancement of sensitivity leading to detectable signals in the picogram range can be obtained.
Düring
K
K
University of Hamburg, Center for Applied Plant Molecular Biology, Federal Republic of Germany.
D000218:Adamantane / Q000031:analogs & derivatives; D015152:Blotting, Northern; D015139:Blotting, Southern; D003124:Colorimetry; D004247:DNA / Q000032:analysis*; D004076:Digoxigenin; D005818:Genetic Engineering / Q000379:methods; D007211:Indoles; D008163:Luminescent Measurements; D008832:Microchemistry / Q000379:methods; D009580:Nitroblue Tetrazolium; D010940:Plant Proteins / Q000032:analysis*; D030821:Plants, Genetically Modified / Q000737:chemistry*; D012313:RNA / Q000032:analysis*
D007211:Indoles; D010940:Plant Proteins; C061523:3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane; D009580:Nitroblue Tetrazolium; C035455:5-bromo-4-chloro-3-indoxyl phosphate; D012313:RNA; D004247:DNA; D004076:Digoxigenin; D000218:Adamantane