Optimization of a digoxigenin-based immunoassay system for gene detection in Arabidopsis thaliana.
Digoxigenin is derived from a plant steroid hormone digoxin found in the plants Digitalis sp. Digoxigenin has been used successfully in labeling nucleic acids. In this experiment we optimized minimum probe requirement for a nonradioactive digoxigenin-based gene detection system in the model plant Arabidopsis thaliana. We showed that 1 microL of labeled probe was sufficient to hybridize onto 1-10 microg of target plasmid DNA. We also examined the sensitivity of labeled probe and showed that 2 microL of labeled probe was not able to hybridize with 1 microg of target DNA, although 2 microL of labeled probe was able to detect target DNA ranging from 2 to 10 microg. To test the efficacy of our optimization protocol, we used 1 microL of labeled plasmid DNA pU16893 harboring an Arabidopsis housekeeping gene elongation factor-1 and showed that the elongation factor-1 gene could be detected in Arabidopsis genome under various environmental conditions. This paper describes a nonradioactive in situ hybridization technique to detect nucleic acids in plants.
Hart
Shanion M
SM
School of Biological Sciences, University of Northern Colorado, Greeley, CO 80639, USA.
Basu
Chhandak
C
D017360:Arabidopsis / Q000235:genetics*; D015342:DNA Probes; D018744:DNA, Plant; D004076:Digoxigenin; D017343:Genes, Plant; D018745:Genome, Plant; D007118:Immunoassay / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D012680:Sensitivity and Specificity
D015342:DNA Probes; D018744:DNA, Plant; D020648:Peptide Elongation Factor 1; D004076:Digoxigenin