Alkaline phosphatase inhibitors as labels of DNA probes.
A new approach to nucleic acid labeling was developed by preparing bifunctional reagents containing, in addition to the DNA-linking group, a competitive inhibitor of the chromogenic enzyme alkaline phosphatase. The nucleic acids labeled in such a way were able to bind themselves to the enzyme, whose activity was restored in the presence of a chromogenic substrate. Five phosphonic-acid-containing reagents were synthesized and coupled to linearized pBR322 plasmid DNA by different condensation methods. Eight probes thus obtained were assayed in a modified dot-blot detection procedure obtaining the best nucleic acid detection sensitivity of 25 pg. Finally, five of the above probes were tested in hybridization experiments, reaching sensitivity of 50 pg.
Davini
E
E
Di Leo
C
C
Rossodivita
A
A
Zappelli
P
P
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors*; D002863:Chromogenic Compounds / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D063065:Organophosphonates / Q000737:chemistry*; D010957:Plasmids / Q000235:genetics; D012680:Sensitivity and Specificity
D002863:Chromogenic Compounds; D015342:DNA Probes; D004791:Enzyme Inhibitors; D063065:Organophosphonates; D000469:Alkaline Phosphatase