title: Characterization of alkaline phosphatase labeled UidA(Gus) probe and its application in testing of transgenic tritordeum.
abstract: Hybridization is a very important molecular biology technique to measure the degree of genetic similarity between DNA sequences, and detect the foreign genes in transgenic organisms. To label a DNA or RNA probe plays a key role in hybridization. A method using nonradioactive material alkaline phosphatase to label UidA(Gus) DNA as probe has been studied. On that basis of Renz and our previous work, alkaline phosphatase-labeled DNA was used as a probe to examine the transformation of the foreign UidA(Gus) gene in transgenic tritordeum. Such DNA-enzyme complexes were characterized and examined carefully, the results showed that it was a sensitive, specific, safe and economical probe. For dot hybridization and Southern blot under full-stringency conditions with alkaline phosphatase as the detector and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-Nitro Blue Tetrazolium (NBT) as the substrate, dot hybridization showed that the UidA(Gus) gene was transformed into the target plants and inherited stable, Southern blot showed that at least two copies of UidA(Gus) gene were inserted into one line of our transgenic tritordeum. Histochemical staining with X-Gluc of transgenic tritordeum also certified that the foreign UidA(Gus) DNA were transformed into the transgenic tritordeum.
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, China-UK Joint Lab, School of life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, China. tuzhiming@hust.edu.cn
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Tu
authors.forename: Jiangzhou
authors.initials: J
authors.lastname: Zhang
authors.forename: Guangxiao
authors.initials: G
authors.lastname: Yang
authors.forename: Guangyuan
authors.initials: G
authors.lastname: He
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D015139:Blotting, Southern; D003116:Color; D003432:Cross-Linking Reagents; D015342:DNA Probes / Q000378:metabolism*; D004795:Enzyme Stability; D005821:Genetic Techniques; D005966:Glucuronidase / Q000378:metabolism*; D006801:Humans; D009693:Nucleic Acid Hybridization; D009928:Organ Specificity; D030821:Plants, Genetically Modified; D006109:Poaceae / Q000235:genetics*; D011058:Pollen / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012639:Seeds / Q000235:genetics; D013194:Staining and Labeling; D013696:Temperature; D013997:Time Factors
chemical_list: D003432:Cross-Linking Reagents; D015342:DNA Probes; D000469:Alkaline Phosphatase; D005966:Glucuronidase