Novel nonisotopic gibberellin DNA probe labeling and fluorescent detection system

摘要

A novel highly sensitive nonisotopic DNA labeling with Gibberellin (GA4) and a new fluorescent fast detection system have been developed. The DNA is labeled with GA4 linked to dUTP via an 8 atom linker spacer (GA4-[8]-dUTP) by a random-primed method. Following hybridization of a membrane-bound target DNA with a GA4-labeled probe, the hybrids are detected by the ELISA principle using anti-GA4 Fab fragments conjugated to alkaline phosphatase (ALP). The novel GA4 system allows the detection of 0.1 pg homologous DNA within several hours in dot- and Southern-blots on nylon membranes using a new fluorochrome, 2-hydroxy-3-naphthoic-acid-2'-phenyl-anilide phosphate (HNPP), as the substrate for ALP. The newly developed GA4 system and HNPP fluorescent detection method exhibits not only high sensitivity and low background, but the sensitivity of DNA detection is identical over a wide range of ratio of GA4-dUTP to dUTP in labeling reaction.